JP7551760B2 - Mammalian Cell Culture Process - Google Patents

Description

The present invention relates to the field of cell culture and the production of recombinant proteins or recombinant viruses in mammalian cells. In particular, the present invention relates to a novel feed medium providing lactate and high concentrations of cysteine, as well as a method for culturing mammalian cells or for producing a product of interest, such as a heterologous protein or a recombinant virus, using said feed medium.

Background of the invention The majority of recombinant therapeutic proteins in the biopharmaceutical industry are produced by mammalian cell culture due to their ability of correct protein folding and post-translational modification. Within mammalian culture systems, Chinese Hamster Ovary (CHO) cells are the host of choice for industrial production processes. Their main advantage is their human-like post-translational modification patterns. In addition, CHO cells have already proven to be safe hosts and are likely to be approved for the manufacture of novel therapeutics. Although the development of stable CHO cell lines with high productivity producing high-quality products has been well-done in the past few years, further improvements in cell culture performance are constantly required. However, other mammalian cell lines such as HEK293, NSO and BHK21 can also be used in the biopharmaceutical industry for protein expression or virus production.

A major strategy for process development is medium design, since cells are constantly interacting with their environment. Growth, productivity, and product quality are directly affected by the choice and composition of the medium used. Optimization of cell culture media to meet cellular demand for nutrients and minimize accumulation of inhibitory substances has many impacts on process performance.

Not only must the composition of the medium be closely observed, but a deeper understanding of the metabolism of the cells is equally crucial for medium development. The metabolism of CHO cells and other mammalian cells is characterized by inefficient and high uptake of substances such as carbon and nitrogen sources, which lead to high concentrations of cytotoxic or inhibitory by-products, such as lactate, ammonia, and various other growth-inhibiting metabolites. Cytotoxic or inhibitory by-products are particularly problematic in fed-batch processes because the medium is not exchanged and thus cytotoxic by-products accumulate over time.

Aiming to overcome such negative characteristics of mammalian cells, and especially CHO cells, numerous strategies have been developed and applied, influencing process parameters such as pH, temperature or _pCO2 . Furthermore, complex feeding strategies and cell line engineering are applied to reduce the formation of inhibitory compounds.

In recent years, bioprocesses using mammalian cells for the production of biopharmaceuticals have been well developed, focusing mainly on improved growth, productivity and product quality. The use of high cell seeding densities (SCD) avoids non-productive growth phases of the cells, resulting in improved space-time yields. However, the demand for optimized nutrient supply to the cells by adjusting the medium design is even more pronounced for cultures with high cell seeding densities, since such cultures are more prone to accumulate potentially inhibitory or cytotoxic metabolites that may result in reduced viability, especially towards the end of the process.

Therefore, there is an ever-increasing demand for further development of stable media that support the growth of high-density mammalian cell cultures and the simultaneous production of large amounts of protein. Historically, media for the culture of animal cells contained plasma, serum, or tissue extracts, which led to unstable and highly variable culture processes due to the high diversity and unclear composition of these complex media components and the inherent risk of viral contamination. Since then, the use of chemically defined serum-free media containing only certain compounds has increased and is the standard in the biopharmaceutical industry today.

Cell culture media consist mostly of energy sources, e.g. carbohydrates or amino acids, lipids, vitamins, trace elements, salts, growth factors, polyamines, and non-nutrient components, e.g. buffers, surfactants, or antifoam agents. Media used in fed-batch culture can be divided into two subgroups: process media (P media), i.e. basal media, and feed media (F media). Basal media contain all essential components at initial concentrations and are used for inoculation. Feed media mostly provide high concentrations of nutrients during the process. Cell culture media are therefore complex compositions of many different compounds, and it is a challenge to identify compounds that result in improved growth, productivity, or product quality. One of the main challenges in media development in recent years is the realization of media that are applicable to different cell lines cultured under different conditions. Media are used under the assumption that the cell lines used are derived from a common host with a common expression vector, which means that they have similar requirements in terms of nutrient supply. This approach allows for rapid process development by shortening timelines and avoiding cell line-specific adjustments of the media. The design of the medium also has a large impact on the critical quality attributes of the desired molecule upstream of production, which poses an additional challenge for medium development, given the wide variety of biopharmaceuticals on the market or in development. In addition to the complexity of the medium design, different culture techniques imply different requirements for the selection of medium composition. The selection of an optimized feeding strategy or type of process, e.g. in perfusion supporting high viable cell density (VCD), has different requirements for supplementation. Thus, cell culture media are complex compositions of many different compounds, and it is a challenge to identify compounds that result in improved growth, productivity, or product quality.

Cysteine is a typical component of mammalian cell culture media. It is not considered to be an essential amino acid, but is nevertheless considered to be a critical amino acid for cell culture and protein synthesis. It is known in the art that insufficient cysteine levels result in reduced protein titers. In particular, insufficient cysteine levels in the feed can result in depletion of cysteine in the cells. This depletion negatively impacts antioxidant molecules such as glutathione (GSH) and taurine, resulting in oxidative stress with multiple deleterious cellular effects. Cysteine is known to be an essential component of cell culture media, however, feeding higher concentrations of cysteine can result in improper disulfide bond pairing and increased protein aggregation in the extracellular environment (Ali A. S., et al., Biotechnol. J., 2019, 14: 1800352).

Lactate, on the other hand, is known as an undesirable by-product with detrimental effects on cell growth and viability. High levels of lactate have been reported to have a clear negative effect on cell culture processes, and therefore attempts have been made to reduce lactate accumulation and/or induce lactate consumption in later culture stages (Li J., et al., Biotechnol Bioeng, 2012, 109(5): p 1173-1186). Thus, as described in the prior art, lactate accumulation or lactate production in mammalian cell cultures was avoided by media containing a combination of asparagine and acidic cystine (WO 2006/026408, e.g. Example 10, FIG. 42), but lactate in particular was not reduced, but rather considered a waste product and was therefore not added or fed to the cell culture. Li et al. used lactate as an alternative feedback pH control strategy and showed for the first time that under lactate-consuming metabolic conditions, exogenous lactate feeding can provide process benefits, in particular reduced ammonium and lower carbon dioxide levels. Ammonia is a by-product of amino acid metabolism and has a negative effect on cell growth. EP 2135946A1 and other state of the art documents such as Kishishita et al., J. Biosci Bioeng. (2015), 120 (1), 78-84, among others, disclose cell culture processes using culture media containing lactate, but explicitly teach that this is considered an undesirable waste product that should be avoided or kept at low concentrations (see paragraph [0046] of EP 2135946A1 and Kishishita et al., p. 81, left column, second paragraph, lines 1-3).

Ritacco F.V. et al, Biotechnol. Prog., 2018, 34(6): 1407-1426, reviews several approaches to the development of CHO cell culture media. It discloses in paragraphs 1408-1410 that lactate, as a product of glucose consumption, can inhibit cell growth in mammalian cell culture. Respective analyses show that in exponential growth phase, CHO cells generate energy mainly via aerobic glycolysis and produce lactate regardless of oxygen concentration, whereas stationary cells mostly perform oxidative phosphorylation and consume lactate. However, in general it can be derived from this disclosure that lactate should be avoided. Furthermore, elevated asparagine concentrations can be useful to reduce lactate and ammonium (page 1412, left column, fourth paragraph, lines 18-20), but the role of cysteine in this context is not discussed.

In view of the ever-increasing demand for further improved methods for culturing mammalian cells in fed-batch cultures for the production of high yields of biopharmaceuticals, including heterologous proteins and recombinant viruses, with high product quality, there remains a need for improved cell culture media and methods using said cell culture media. It is therefore an object of the present invention to provide an improved fed-batch method for the production of a product of interest in mammalian cells.

SUMMARY OF THEINVENTION The present invention relates to the surprising combined effect of lactate and cysteine on cell culture performance and/or product quality.

In one aspect, a method is provided for producing a product of interest in a fed-batch process comprising the steps of: (a) providing mammalian cells comprising a nucleic acid encoding a product of interest; (b) inoculating a basal medium with the mammalian cells to provide a cell culture; (c) adding a feed medium to the cell culture comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium or to the resulting cell culture medium with lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; (d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest; and (e) optionally isolating the product of interest. Preferably, the feed medium is added once a day, more preferably continuously. The product of interest is preferably a heterologous protein or a recombinant virus and/or the basal medium and the feed medium are preferably serum-free and chemically defined. In certain preferred embodiments, the lactate/cysteine molar ratio is from about 10:1 to 50:1, preferably from about 10:1 to about 30:1.

Lactate can be added at 3 mmol/L/day or more, 5 mmol/L/day or more, 7 mmol/L/day or more, or 10 mmol/L/day or more. In certain embodiments, lactate in the cell culture medium is maintained at 0.5 g/L or more, 1 g/L or more, 2 g/L or more, preferably 2 to 4 g/L.

Cysteine may be provided as cysteine or its salts and/or hydrates, cystine or its salts, or a dipeptide or tripeptide containing cysteine. Regardless of the form in which it is provided, cysteine may be added at 0.25 mM/day or more, 0.3 mM/day or more, or 0.4 mM/day or more.

In certain embodiments, the nucleic acid encodes a heterologous protein and the product titer and/or specific productivity of the cells is increased compared to the product titer and/or specific productivity of the cells of a heterologous protein produced by the same method, wherein the feed medium is supplemented with cysteine at 0.19 mM/day or less in the absence of lactate. Alternatively or additionally, the nucleic acid encodes a heterologous protein and the relative amount of high mannose structures in the population of heterologous proteins is decreased compared to the population of heterologous proteins produced by the same method, wherein the feed medium is supplemented with cysteine at 0.19 mM/day or less in the absence of lactate. Preferably, the high mannose structures are structures with five mannose units. Alternatively or additionally, the nucleic acid encodes a heterologous protein and the relative amount of acidic species (relative to the total) in the population of heterologous proteins is decreased compared to the population of heterologous proteins produced by the same method, wherein the feed medium is supplemented with cysteine at the same concentration in the absence of lactate.

The heterologous protein is preferably an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. In one embodiment, the antibody, bispecific antibody, or trispecific antibody is an IgG1 antibody, an IgG2a antibody, an IgG2b antibody, an IgG3 antibody, or an IgG4 antibody.

Also provided is a method for culturing mammalian cells in a fed-batch process comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a product of interest; b) inoculating the mammalian cells into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium, lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; and d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest.

In another aspect, the method comprises the steps of: a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cell into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium, in a molar ratio of lactate/cysteine of about 8:1 to about 50:1 (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ a) adding lactate and cysteine at least at 0.225 mM/day; d) culturing mammalian cells in a cell culture medium under conditions that permit expression of the heterologous protein; and e) optionally isolating the heterologous protein; wherein the relative amount of acidic species in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium has been supplemented with the same concentration of cysteine in the absence of lactate.

In yet another aspect, the method comprises the steps of: a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cell into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium or to the resulting cell culture medium, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium comprising adding one or more feed supplements to the resulting ... resulting cell culture medium, the feed medium comprising adding one or more feed supplements to the resulting cell culture medium, the feed medium ^comprising adding one ^or more feed supplements to the resulting cell culture ^{medium ,} a) adding lactate and cysteine at 0.225 mM/day or more; d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the heterologous protein; wherein the relative amount of high mannose structures in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium is added with 0.19 mM/day or less of cysteine in the absence of lactate, and preferably wherein the high mannose structures are structures with 5 mannose units.

In yet another aspect, a method for producing a heterologous protein comprises the steps of: (a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; (b) inoculating the mammalian cell into a basal medium to provide a cell culture; and (c) adding one or more feed supplements to the cell culture, the feed medium comprising a lactate/cysteine molar ratio of about 8:1 to about 50:1 (mmol×L− ¹ ×day−1/mmol×L ^{− 1} ×day ⁻¹⁾ to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium. (d) culturing mammalian cells in a cell culture medium under conditions that permit expression of the heterologous protein; and (e) optionally isolating the heterologous protein from the mammalian cells; wherein the negative effect of cysteine on product quality characteristics in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium has been supplemented with the same concentration of cysteine in the absence of lactate.

The mammalian cells used in the methods of the present invention can be any mammalian cell or cell line, preferably the mammalian cells are HEK293 cells or CHO cells, or cells derived from HEK293 cells or CHO cells, preferably the mammalian cells are CHO cells or CHO-derived cells.

Also provided is a heterologous protein produced by any of the methods described in the present invention, preferably produced in a fed-batch process as described herein, by a method for reducing acidic species in the heterologous protein or reducing high mannose structures in the heterologous protein. The heterologous protein may also be produced by a method that prevents the negative effect of cysteine on product quality characteristics when producing the heterologous protein as described herein.

In yet another aspect, the present invention relates to the use of lactate in a feed medium to reduce acidic species in a heterologous protein produced in a fed-batch process, wherein the feed medium is supplemented with cysteine at 0.225 mM/day or more.

Also provided is the use of lactate in a feed medium to reduce high mannose structures in a heterologous protein produced in a fed-batch process, wherein the feed medium comprises cysteine at 0.225 mM/day or more. Preferably, the high mannose structures are structures having 5 mannoses.

Also provided is the use of lactate in a feed medium to prevent the negative effects of cysteine on product quality characteristics of a heterologous protein produced in a fed-batch process, preferably wherein the negative effects on product quality characteristics are increased high mannose structures, increased low molecular weight species, and/or increased acidic species.

There is also provided the use of lactate and cysteine in a feed medium to increase the titer of a heterologous protein and/or the specific productivity of a cell in a fed-batch process. Preferably, the fed-batch process comprises culturing mammalian cells, wherein the mammalian cells are preferably HEK293 cells or CHO cells or cells derived from HEK293 cells or CHO cells, preferably the mammalian cells are CHO cells or CHO-derived cells.

In yet another aspect, a feed medium for fed-batch culture of mammalian cells is provided, comprising lactate and cysteine in a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1. Preferably, the feed medium includes one or more feed supplements for separate addition.

In yet another aspect, a kit is provided that includes (a) a concentrated feed medium for fed-batch culture of mammalian cells, the concentrated feed medium including lactate and optionally cysteine, and (b) aqueous supplements segregated from the concentrated feed medium including cysteine, where the feed medium and supplements provide a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1 and 0.225 mM/day or more of cysteine, with daily addition of less than 5%, preferably less than 3.5%, of the starting cell culture volume.

Viable cell density (A), viability (D), relative IgG titer (C), and lactate concentration (D) of the ultra-high seeding density (uHSD) process using a seeding concentration of ^10x106 cells/ml in a 3L bioreactor. CHO cells were in culture for 13-14 days using a routine ultra-high seeding density in the presence or absence of bolus addition of lactate and/or cysteine. (C) IgG concentration on the y-axis is given as titer relative to the highest measured value (100%). Viable cell density (A), viability (D), relative IgG titer (C), and lactate concentration (D) of the ultra-high seeding density (uHSD) process using a seeding concentration of ^10x106 cells/ml in a 3L bioreactor. CHO cells were in culture for 13-14 days using a routine ultra-high seeding density in the presence or absence of bolus addition of lactate and/or cysteine. (C) IgG concentration on the y-axis is given as titer relative to the highest measured value (100%). Viable cell density (A), viability (D), relative IgG titer (C), and lactate concentration (D) of the ultra-high seeding density (uHSD) process using a seeding concentration of ^10x106 cells/ml in a 3L bioreactor. CHO cells were in culture for 13-14 days using a routine ultra-high seeding density in the presence or absence of bolus addition of lactate and/or cysteine. (C) IgG concentration on the y-axis is given as titer relative to the highest measured value (100%). Viable cell density (A), viability (D), relative IgG titer (C), and lactate concentration (D) of the ultra-high seeding density (uHSD) process using a seeding concentration of ^10x106 cells/ml in a 3L bioreactor. CHO cells were in culture for 13-14 days using a routine ultra-high seeding density in the presence or absence of bolus addition of lactate and/or cysteine. (C) IgG concentration on the y-axis is given as titer relative to the highest measured value (100%). Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viable cell density, viability, product titer, and lactate concentration of two cell line experimental design experiments of a routine process in 250 ml bioreactors. (A, B, C, and D) Cell cultures of cell line A (CHO-K1; IgG1) were cultured with 0 g/L/day sodium lactate and 0 ml/L/day cystine (0 Lac/0 Cystine; control), 30 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day secondary cystine (30 Lac/0.84 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day secondary cystine (15 Lac/1.67 Cystine). (A) Viable cell density (10 x ¹⁰⁶ cells/ml), (B) Viability [%], (C) IgG titer [%] compared to the highest measured value, and (D) Lactate concentration [g/L] are provided. (E, F, and G) Cell cultures of cell line B (CHO-K1; IgG4) were cultured with 0 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (0 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 1.67 ml/L/day 2nd cystine (30 Lac/1.67 Cystine), 30 g/L/day sodium lactate fed at 17.2 g/L and 0 ml/L/day 2nd cystine (30 Lac/0 Cystine), and 15 g/L/day sodium lactate fed at 17.2 g/L and 0.84 ml/L/day 2nd cystine (15 Lac/0.84 Cystine). (E) Viable cell density [10 x ¹⁰⁶ cells/ml], (F) Viability [%], (G) IgG titer [%] compared to the highest measured value, and (H) Lactate concentration [g/L] are provided. Viability of cell line A harvest as a function of lactate and cystine feeding ( ^R2 : 0.95; ^Q2 : 0.85). The unit g/L on the y-axis refers to the addition of sodium lactate; the unit ml/L/day on the x-axis refers to the addition of 17.2 g/L of cystine. Product titer of cell line A as a function of lactate and cystine feeding ( ^R2 : 0.98; ^Q2 : 0.96). The units g/L on the x-axis refer to sodium lactate addition; the units ml/L/day on the y-axis refer to 17.2 g/L cystine addition. The highest product titer could be obtained with high lactate and high cystine feeding. Normalized titer values (%) are normalized to the highest value (among the cell lines used in the experimental design) of the experimental design. Acidic peak profile (APG) of cell line A as a function of lactate and cystine feeding ( ^R2 : 0.98; ^Q2 : 0.97). The units g/L on the y-axis refer to the addition of sodium lactate; the units ml/L/day on the x-axis refer to the addition of 17.2 g/L of cystine. The increase in APG due to cystine feeding can be strongly reduced through additional lactate feeding. Viability of cell line B harvest as a function of lactate and cystine feeding ( ^R2 : 0.95; ^Q2 : 0.85). The unit g/L on the y-axis refers to the addition of sodium lactate; the unit ml/L/day on the x-axis refers to the addition of 17.2 g/L of cystine. Product titer of cell line B as a function of lactate and cystine feeding ( ^R2 : 0.98; ^Q2 : 0.96). The units g/L on the x-axis refer to sodium lactate addition; the units ml/L/day on the y-axis refer to 17.2 g/L cystine addition. The highest product titer could be obtained with high lactate and high cystine feeding. Normalized titer values (%) are normalized to the highest value (among the cell lines used in the experimental design) of the experimental design. Acidic peak profile (APG) of cell line B as a function of lactate and cystine feeding ( ^R2 : 0.98; ^Q2 : 0.97). The units g/L on the y-axis refer to the addition of sodium lactate; the units ml/L/day on the x-axis refer to the addition of 17.2 g/L of cystine. The increase in APG due to cystine feeding can be strongly reduced through additional lactate feeding. Figure 1. Pentamannose structures of cell line A (A) and cell line B (B) as a function of lactate ( ^R2 : 0.93; ^Q2 : 0.77). The units g/L on the x-axis refer to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Figure 1. Pentamannose structures of cell line A (A) and cell line B (B) as a function of lactate ( ^R2 : 0.93; ^Q2 : 0.77). The units g/L on the x-axis refer to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Low molecular weight species (LMW) of two cell lines as a function of cystine and lactate. (A and B) Low molecular weight species (LMW) of cell line A as a function of (A) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (B) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. (C and D) Low molecular weight species (LMW) of cell line B as a function of (C) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (D) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Normalized LMW values (%) on the y-axis are normalized to the highest value of the experimental design. Low molecular weight species (LMW) of two cell lines as a function of cystine and lactate. (A and B) Low molecular weight species (LMW) of cell line A as a function of (A) cystine shown as ml/L/day on the x-axis referring to the addition of 17.2 g/L cystine, and (B) lactate shown as g/L on the x-axis referring to the addition of sodium lactate. (C and D) Low molecular weight species (LMW) of cell line B as a function of (C) cystine shown as ml/L/day on the x-axis referring to the addition of 17.2 g/L cystine, and (D) lactate shown as g/L on the x-axis referring to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Normalized LMW values (%) on the y-axis are normalized to the highest value of the experimental design. Low molecular weight species (LMW) of two cell lines as a function of cystine and lactate. (A and B) Low molecular weight species (LMW) of cell line A as a function of (A) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (B) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. (C and D) Low molecular weight species (LMW) of cell line B as a function of (C) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (D) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Normalized LMW values (%) on the y-axis are normalized to the highest value of the experimental design. Low molecular weight species (LMW) of two cell lines as a function of cystine and lactate. (A and B) Low molecular weight species (LMW) of cell line A as a function of (A) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (B) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. (C and D) Low molecular weight species (LMW) of cell line B as a function of (C) cystine shown as ml/L/day on the x-axis, which refers to the addition of 17.2 g/L of cystine, and (D) lactate shown as g/L on the x-axis, which refers to the addition of sodium lactate. Confidence intervals (95%) are presented as dotted lines. Normalized LMW values (%) on the y-axis are normalized to the highest value of the experimental design. Shown are the viable cell density (A), viability (B), lactate concentration (C), and relative IgG titer (D) of cell line C. Cell cultures were supplemented with 14.37 g/L cystine in an additional feed at 2 ml/L/day (w Cys) or 30 g/L lactate with no feed medium at 30 ml/L/day (w Lac), or both (w Cys/Lac). Control cells were fed with feed medium only (feed 1). Shown are the viable cell density (A), viability (B), lactate concentration (C), and relative IgG titer (D) of cell line C. Cell cultures were supplemented with 14.37 g/L cystine in an additional feed at 2 ml/L/day (w Cys) or 30 g/L lactate with no feed medium at 30 ml/L/day (w Lac), or both (w Cys/Lac). Control cells were fed with feed medium only (feed 1). Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line D after treatment as described in the figure legend of FIG. Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line D after treatment as described in the figure legend of FIG. Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line E following treatment as described in the figure legend of FIG. Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line E following treatment as described in the figure legend of FIG. Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line F after treatment as described in the figure legend of FIG. Shown are viable cell density (A), viability (B), lactate concentration (C), and relative IgG titers (D) of cell line F after treatment as described in the figure legend of FIG. Figure 2. Product titer of cell line A as a function of lactate and cystine feeding in the design of experiment optimization of the ultra-high seeding density process (R2: 0.84; Q2: 0.77). The highest product titer could be obtained with high lactate and high cysteine feeding. Normalized titer values (%) are normalized to the highest value of the design of experiment (among the cell lines used in the design of experiment). Figure 2. Product titer of cell line B as a function of lactate and cystine feeding in a design of experiment optimization of the ultra-high seeding density process (R2: 0.84; Q2: 0.77). The highest product titer could be obtained with high lactate and high cysteine feeding. Normalized titer values (%) are normalized to the highest design of experiment value (among the cell lines used in the design of experiment). Viability of the cell line A collection as a function of lactate feeding (R2: 0.94; Q2: 0.89). Confidence intervals (95%) are presented as dotted lines. Viability of the cell line B collection as a function of lactate feeding (R2: 0.94; Q2: 0.89). Confidence intervals (95%) are presented as dotted lines. Acidic peak variants (APG) of cell line A as a function of lactate and cystine feeding (R2: 0.99; Q2: 0.97). The increase in APG due to cysteine feeding can be strongly reduced through additional lactate feeding. Acidic peak variants (APG) of cell line B as a function of lactate and cystine feeding (R2: 0.99; Q2: 0.97). The increase in APG due to cysteine feeding can be strongly reduced through additional lactate feeding. Figure 2: Pentamannose structure (Man5) of cell line A as a function of lactate (R2: 0.71; Q2: 0.48). Confidence intervals (95%) are presented as dotted lines. Figure 2: Pentamannose structure (Man5) of cell line B as a function of lactate (R2: 0.71; Q2: 0.48). Confidence intervals (95%) are presented as dotted lines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The general embodiments "comprising" or "comprised" encompass the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting sense. As used herein, the singular forms "a,""an," and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

The term "cell culture" or "cell culture" includes the process of cell culture and fermentation at all scales (e.g. from microtiter plates to large industrial bioreactors, i.e. from sub-mL scale to over 10,000 L), in all different process modes (e.g. batch culture, fed-batch culture, perfusion culture, continuous culture), in all process control modes (e.g. uncontrolled, fully automated and controlled systems with e.g. pH, temperature, oxygen content control), in all types of fermentation systems (e.g. single-use systems, stainless steel systems, glassware systems). According to the present invention, the cell culture is a mammalian cell culture and is a fed-batch culture. In a preferred embodiment, the cell culture is a cell culture with a volume of more than 1 L, preferably more than 2 L, more than 10 L, more than 1,000 L, more than 5000 L, more preferably more than 10,000 L.

The term "fed-batch" as used herein refers to a cell culture in which the cells are continuously or periodically fed with a nutrient-containing feed medium. Feeding can begin on day 0 immediately after the start of the cell culture, or more typically on days 1, 2, or 3 after the start of the culture. Feeding can follow a predefined schedule, such as daily, every 2 days, every 3 days, etc. Alternatively, the culture can be monitored for cell growth, nutrients, or toxic by-products, and feeding can be adjusted accordingly. In general, the following parameters are often determined on a daily basis, covering viable cell concentration, product concentration (titer), and several metabolites, such as glucose, pH, lactate, osmolality (a measure of salt content), and ammonium (a growth inhibitor that negatively affects the growth rate and reduces viable biomass). Higher product titers can be reached in fed-batch mode compared to batch cultures (cultures without feeding). Typically, the fed-batch culture is stopped at some point, the cells and/or medium are harvested, and the product of interest, e.g., a heterologous protein or recombinant virus, is isolated and/or purified. Fed-batch processes are typically maintained for about 2-3 weeks, e.g., about 10-24 days, about 12-21 days, about 12-18 days, preferably about 12-16 days. In particular, fed-batch processes for the production of heterologous proteins are typically maintained for about 2-3 weeks, e.g., about 10-24 days, about 12-21 days, about 12-18 days, preferably about 12-16 days.

For recombinant virus production, cells are typically transduced with the recombinant virus at a desired cell density. Feeding can be initiated on day 0 immediately after the initiation of cell culture, or more typically on days 1, 2, or 3 after the initiation of culture, where cells are transduced with the recombinant virus at the desired cell density at the time of cell inoculation or after a certain period of time when the desired cell density is reached (which can be, for example, days 1-7 after the initiation of culture, preferably days 2-5 after the initiation of culture, more preferably days 3-5 after the initiation of culture after feeding has begun). Alternatively, cells may be stably transfected with one or more nucleic acid molecules encoding the recombinant virus, or cells may be transiently transfected with one or more nucleic acid molecules encoding the recombinant virus, or a combination thereof. Similar to viral transduction of mammalian cells, in transient transfection, cells can be transfected with one or more nucleic acids encoding a recombinant virus at the time of cell inoculation or after a certain period of time when the desired cell density is reached (this can be after feeding has commenced, e.g., 1-7 days after the start of culture, preferably 2-5 days after the start of culture, more preferably 3-5 days after the start of culture) at the desired cell density.

By definition, any nucleic acid, sequence, or gene introduced into a host cell is called a "heterologous nucleic acid," "heterologous sequence," "heterologous gene," "heterologous RNA" or "transgene" or "recombinant gene" to the host cell, even if the introduced sequence is identical to an endogenous nucleic acid, sequence, or gene in the host cell. Thus, a "heterologous" or "recombinant" protein or RNA is a protein or RNA expressed from a heterologous nucleic acid, sequence, or gene, preferably DNA. In a preferred embodiment, the introduced nucleic acid, sequence, or gene is not identical to an endogenous nucleic acid sequence or gene of the host cell in question.

The term "encode" or "encoding" as used herein broadly refers to any process that uses information within a polymeric macromolecule to direct the production of a second molecule that is different from a first molecule. The second molecule may have a different chemical structure than the chemical nature of the first molecule. For example, in some aspects, the term "encode" describes a semi-conservative DNA replication process in which one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by a transcription process using a DNA-dependent RNA polymerase enzyme). An RNA molecule can also encode a polypeptide, as in a process called translation. When used to describe the translation process, the term "encode" also extends to triplet codons that code for amino acids. In some aspects, an RNA molecule can encode a DNA molecule, for example, by a reverse transcriptase process that incorporates an RNA-dependent DNA polymerase. In another aspect, the DNA molecule can encode a polypeptide, where "encode" as used herein is understood to encompass both the processes of transcription and translation.

The terms "polypeptide" and "protein" are used synonymously. These terms refer to polymers of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions including, but not limited to, glycosylation, glycation, acetylation, phosphorylation, oxidation, amidation, or protein processing. Modifications and changes, such as fusion to other proteins, substitutions, deletions, or insertions of amino acid sequences, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, substitutions of specific amino acid sequences can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with similar or modified properties. Amino acid modifications can be prepared, for example, by performing site-directed mutagenesis or polymerase chain reaction-mediated mutagenesis on the underlying nucleic acid sequence. Thus, the terms "polypeptide" and "protein" also include, for example, fusion proteins consisting of an immunoglobulin component (e.g., Fc component) and a growth factor (e.g., an interleukin), an antibody, or any antibody-derived molecular format or antibody fragment.

As used herein, the term "product of interest" refers to any product produced in a mammalian cell, particularly heterologous proteins and recombinant viruses.

As used herein, the term "cell culture medium" refers to a medium for culturing mammalian cells that contains minimum essential nutrients and components, such as vitamins, trace elements, salts, bulk salts, amino acids, lipids, carbohydrates, preferably in a buffered medium. Typically, cell culture media for mammalian cells have a near neutral pH, e.g., a pH of about 6.5 to about 7.5, preferably about 6.8 to about 7.3, more preferably about 7. Non-limiting examples of such cell culture media include commercially available media such as Ham F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (IMDM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Improved Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S (Invitrogen), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma), as well as proprietary media from various sources. The cell culture medium may be a basal cell culture medium. The cell culture medium may also be a basal cell culture medium to which a feed medium and/or additives have been added. The cell culture medium may also be referred to as a fermentation broth if the cells are cultured in a fermenter or bioreactor.

The term "basal medium" or "basal cell culture medium" as used herein is a cell culture medium for culturing mammalian cells as defined below. It refers to the medium in which cells are cultured from the beginning of the cell culture operation, which is typically not used as an additive to another medium, although various components may be added to the basal medium. The basal medium serves as a base to which, optionally, further additives (or supplements) and/or feed medium may be added during the culture (i.e., the cell culture operation that results in the cell culture medium). The basal cell culture medium is provided from the beginning of the cell culture process. Generally, the basal cell culture medium provides nutrients such as a carbon source, amino acids, vitamins, bulk salts (e.g., sodium chloride or potassium chloride), various trace elements (e.g., manganese sulfate), pH buffers, lipids, and glucose. Major bulk salts are usually provided only in the basal medium, and the final osmolality in the cell culture should not exceed about 280-350 mOsmol/kg, so that the cell culture can grow and proliferate with reasonable osmotic stress.

The term "feed" or "feed medium" as used herein relates to a nutrient concentrate/concentrated nutrient composition used as a feed in mammalian cell cultures. It is therefore provided as a concentrate, which is added to the cell culture. It is provided as a "concentrated feed medium" to minimize dilution of the cell culture, typically the feed medium is provided at 10-50 ml/L/day, preferably 15-45 ml/L/day, more preferably 20-40 ml/L/day, even more preferably 30 ml/L/day, based on the culture starting volume in the vessel (CSV, meaning starting volume on day 0). This corresponds to a daily addition of about 1-5%, preferably about 1.5-4.5%, more preferably about 2-4%, even more preferably about 3% of the culture starting volume. In cultures using high density inoculation or very high density inoculation, higher feeding rates may be beneficial, for example 10-50 ml/L/day, 15-45 ml/L/day, or 25-45 ml/L/day. This corresponds to a daily addition of about 1-5%, about 1.5-4.5%, or about 2.5-4.5% of the culture starting volume. The feeding rate should be understood as the average feeding rate during the feeding period. The feed medium typically has most, if not all, components of the basal cell culture medium at higher concentrations. In general, the feed medium replaces nutrients consumed during cell culture, such as amino acids and carbohydrates, while salts and buffers are less important, which are generally provided together with the basal medium. The feed medium is typically added to the (basal) cell culture medium/fermentation broth in a fed-batch mode. The cell culture medium is obtained by the feed medium added (repeatedly or continuously) to the basal medium. The feed may be added in different modes, such as continuous or bolus addition, or via perfusion-related techniques (chemostat or hybrid perfusion systems). Preferably, the feed medium is added once a day, but it may also be added more frequently, such as twice a day, or less frequently, such as every two days. More preferably, the feed medium is added continuously. Nutrient addition generally occurs during the culture (i.e. after day 0). In contrast to the basal medium, the feed medium typically consists of a highly concentrated nutrient solution (e.g. more _{than 6 - fold} ) that provides all components similar to the basal medium, except for "active compounds with high osmolarity" such as major bulk salts (e.g. NaCl, KCl, NaHCO ₃ , MgSO 4 , Ca(NO 3 ) 2 ). Typically, a 6-fold or greater concentration of the basal medium, without or with reduced bulk salts, maintains good solubility of the compounds and a sufficiently low osmolarity (e.g. 270-1500 mOsmol/kg, preferably 310-800 mOsmol/kg) to maintain an osmolarity in the cell culture of about 270-550 mOsmol/kg, preferably about 280-450 mOsmol/kg, more preferably about 280-350 mOsmol/kg. The feed medium may be added as one complete feed medium or may contain one or more feed supplements for separate addition to the cell culture. The use of one or more feed supplements may be required due to different feeding regimes, such as, for example, periodic feeding and on-demand feeding, which is often implemented for the addition of glucose, and therefore is typically provided as at least a separate feed. The use of one or more feed supplements may also be necessary due to low solubility of certain compounds, solubility of certain compounds at different pHs, and/or interactions of compounds in the feed medium at high concentrations. The feed medium is preferably chemically defined (optionally containing recombinant proteins, such as insulin or insulin-like growth factors). It does not contain cells, is not in contact with cells in culture, or does not contain metabolic waste products derived from cells. Thus, the term "feed medium" as used herein excludes preconditioned medium obtained from a cell culture or culture medium in a cell culture, i.e. in the presence of cells (also referred to herein as cell culture medium).

The term "feed supplement" as used herein relates to a concentrate of nutrients that can be added to the feed medium prior to use or that can be added to the basal medium and/or cell culture medium separately from the feed medium. Thus, a compound can be provided with the feed medium or the feed supplement, or a compound can be provided with the feed medium and the feed supplement. For example, cysteine may be added with the feed medium and the feed supplement in a two-feed strategy. As a feed medium, the "feed supplement" is provided as a concentrate to avoid dilution of the cell culture liquid.

The cell culture medium (both the basal medium and the feed medium) is preferably serum-free and of defined chemical composition. The basal medium and/or the feed medium may furthermore be protein-free. As used herein, "serum-free medium" refers to a cell culture medium for in vitro cell culture, which does not contain serum of animal origin. This is preferred because serum may contain contaminants, e.g. viruses, originating from said animals, and serum is not well defined in composition and varies from batch to batch. The basal medium and the feed medium according to the present invention are serum-free.

As used herein, a "chemically defined medium" refers to a cell culture medium suitable for in vitro cell culture, in which all components are known. More specifically, it does not contain any supplementary substances such as animal serum or plant, yeast or animal hydrolysates. Therefore, a chemically defined medium is also serum-free. The basal medium and the feed medium according to the present invention are preferably chemically defined. In one embodiment, the basal medium and/or the feed medium are serum-free and chemically defined, optionally including recombinant growth factors, such as insulin or insulin-like growth factors (IGFs). The basal medium and/or the feed medium as referred to herein does not contain any other proteins, except for proteins produced by the mammalian cells to be cultured, that were previously present in the cell culture fluid for preparing the cell culture medium.

As used herein, "protein-free medium" refers to a cell culture medium for in vitro cell culture that does not contain any protein (except for proteins produced by the cells once cultured in the cell culture medium), where protein refers to a polypeptide of any length, but excluding single amino acids, dipeptides, or tripeptides. In particular, growth factors, such as insulin and insulin-like growth factors (IGFs), are not present in the medium. Preferably, the basal medium and feed medium described in the present invention are chemically defined and protein-free.

The term "viability" as used herein refers to the percentage of living cells in a cell culture as determined by methods known in the art, for example the trypan blue exclusion method using a Cedex instrument based on an automated microscopic cell count (Innovatis AG, Bielefeld). However, numerous other methods exist for the determination of viability, such as fluorimetric methods (for example based on propidium iodide), calorimetric methods or enzymatic methods used to reflect the energy metabolism of living cells, for example using LDH lactate dehydrogenase or certain tetrazolium salts, such as Alamar Blue, MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) or TTC (tetrazolium chloride).

As used herein, the terms "producing" or "producing in large quantities", "production", "production and/or secretion", "producing", "producing cells" or "producing at high yields" refer to the production of a product of interest encoded by a nucleic acid, e.g., a heterologous protein or a recombinant virus. "Increased production and/or secretion" or "production at high yields" refers to the expression of a product of interest, e.g., a heterologous protein or a recombinant virus, and in the context of a heterologous protein, it means an increase in the specific productivity of the cells, an increased titer, an increased overall productivity of the cell culture, or a combination thereof. In the context of a recombinant virus, it means an increase in cell-specific and/or total particles produced, an increase in cell-specific and/or total infectious particles, or a combination thereof. As used herein, increased titer refers to an increased concentration in the same volume, i.e., an increase in the total yield, which can be used for heterologous proteins as well as recombinant viruses.

As used herein, the terms "enhanced," "enhanced," "enhanced," "increased," or "increased" generally mean an increase of at least about 10% compared to a control cell culture, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or any integer increase between 10 and 300% compared to a control cell culture. As used herein, a "control cell culture" or "control mammalian cell culture" is a cell culture using the same cells (same cell clone) producing the same product using the same method described in the present invention, where the feed medium is supplemented with 0.19 mM/day or less of cysteine in the absence of lactate.

Method for Producing a Product of Interest In one aspect, the present invention relates to a method for producing a product of interest in a fed-batch process comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding the product of interest; b) inoculating a basal medium with the mammalian cells to provide a cell culture; c) adding a feed medium to the cell culture comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium or to the resulting cell culture medium with lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or more; d) culturing the mammalian cells in the cell culture medium under conditions allowing expression of the product of interest; and e) optionally isolating the product of interest. Preferably, the product of interest is a heterologous protein or a recombinant virus, more preferably a heterologous protein.

Also provided is a method of culturing mammalian cells in a fed-batch process comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a product of interest; b) inoculating a basal medium with the mammalian cells to provide a cell culture; c) adding a feed medium to the cell culture comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium, lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; and d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest. Optionally, the product of interest may be further purified or isolated. Preferably, the product of interest is a heterologous protein or a recombinant virus, more preferably a heterologous protein.

The feed medium used in the method according to the invention is added once a day, preferably continuously, during the feeding period of the fed-batch process. In one embodiment, the feed medium is added starting from day 0 to day 5. The skilled person will understand that this may also depend on the seeding density. For normal seeding densities ( ^0.7-1x106 cells/ml), feeding typically starts on day 1-5, preferably on day 2-3. Feeding typically continues until at least 5 days before the end of the fed-batch process, at least 4 days before the end of the fed-batch process, at least 3 days before the end of the fed-batch process, at least 2 days before the end of the fed-batch process, preferably until the end of the process. More preferably, feeding starts on day 2-3 and continues until at least 2 days before the end of the fed-batch process, more preferably until the end of the fed-batch process of the cells. For high seeding densities (greater than 1 to ^4x106 cells/ml), feeding typically begins on days 0 to 4, preferably on days 0 to 2. Feeding typically continues until at least 5 days before the end of the fed-batch process, at least 4 days before the end of the fed-batch process, at least 3 days before the end of the fed-batch process, and may continue until the end of the process. Preferably, feeding begins on days 0 to 2 and continues until at least 4 days or 3 days before the end of the fed-batch process. For very high seeding densities (greater than 4 to ^20x106 cells/ml), feeding typically begins on days 0 to 3, preferably on days 0 to 1. Feeding typically continues until at least 5 days before the end of the fed-batch process, at least 4 days before the end of the fed-batch process, at least 3 days before the end of the fed-batch process, and may continue until the end of the process. Preferably, feeding is started on day 0 and continues until at least 4 or 3 days before the end of the fed-batch process. Thus, depending on the start of the addition of the feed medium, the method may further comprise steps bi) to b) (inoculating the mammalian cells) and c) (adding the feed medium), where step bi) comprises culturing the mammalian cells in a basal medium. The unit "mmol x L ^-1 x day ^-1 " used herein to define the addition of lactate or cysteine may also be referred to as mmol/L/day or mM/day. It refers to mmol/L given per day, regardless of whether the addition is a bolus addition or a continuous addition. According to the present invention, the cells are preferably in a metabolic state to consume lactate in step (c) and/or when lactate is added to the culture.

The term "inoculating" as used herein refers to the process of collecting a sample of mammalian cells, such as a mammalian cell line, and placing them in a medium containing nutrients required for growth. Typically, the mammalian cells are placed in a basal medium for growth or production. This process may also be referred to as seeding. The mammalian cells may be inoculated into the basal medium at different seeding densities. The term "seeding" or "normal seeding" as referred to herein refers to a standard seeding density of about ^0.7-1x10 cells/ml to about ^1x10 cells/ml, the term "high seeding" refers to a seeding density of more than ^1x10 cells/ml to about ^4x10 cells/ml, and the term "ultra-high seeding" refers to a seeding density of more than ^4x10 cells/ml to about ^20x10 cells/ml or even higher, preferably about ^6x10 cells/ml to about ^15x10 cells/ml, more preferably ^8x10 cells/ml to about ^12x10 cells/ml.

The lactate/cysteine molar ratio can be from about 10:1 to about 50:1, preferably from about 10:1 to about 30:1, preferably from about 15:1 to about 30:1.

In one embodiment, lactate is added at 3 mmol/L/day or more, 3.8 mmol/L/day or more, 5 mmol/L/day or more, preferably 7 mmol/L/day or more, 7.8 mmol/L/day or more, 10 mmol/L/day or more, or even 15 mmol/L/day or more.

Lactate in the cell culture medium is maintained at 0.5 g/L or more, 1 g/L or more, preferably 2 g/L or more, preferably 2-4 g/L, more preferably 2-3 g/L. The concentration in the cell culture medium should be maintained below 5 g/L (about 56 mM), at which point lactate becomes toxic. Thus, the concentration of lactate in the cell culture medium is maintained at about 1 g/L to about 4.5 g/L (about 10-50 mM), about 2 g/L to about 4 g/L (about 20-45 mM), preferably about 2 g/L to about 3 g/L (about 20-35 mM). Lactate (molecular weight = 89.07 g/mol) may be provided as its salts, esters, and/or hydrates, and/or lactic acid, preferably as a salt, e.g. sodium lactate (molecular weight = 112.06 g/mol) (where 1 g/L of lactate is equivalent to about 1.25 g/L of sodium lactate). Exemplary esters of lactate are e.g. ethyl lactate or butyl lactate. Lactic acid may also be used in the context of the present invention. However, it may affect the pH of the feed medium, and therefore it is preferably titrated with sodium hydroxide to sodium lactate before addition to or mixing with the components of the feed medium. The lactate salts, esters, and/or hydrates, or lactic acid are provided in equimolar concentrations to the lactate concentrations provided herein. In a preferred embodiment, the basal medium does not contain lactate added as a medium component. However, lactate may be produced in the basal medium as a metabolite during the culture. Lactate salts are obtained as sodium lactate or lactic acid, which is titrated with sodium hydroxide to sodium lactate prior to addition to the feed medium. As used herein, the term "lactate salt" refers to L-lactate salts. Thus, for example, sodium lactate and lactic acid refer to sodium L-lactate and L-lactic acid.

Cysteine may be provided as cysteine or its salts and/or hydrates, cystine or its salts, or a dipeptide or tripeptide containing cysteine. The salts and/or hydrates of cysteine, or cystine or its salts, or a dipeptide or tripeptide containing cysteine are provided in equimolar concentrations to the concentrations of cysteine provided herein. As used herein, the terms "cysteine" and "cystine" refer to L-cysteine and L-cystine. According to the present invention, cysteine is added at 0.225 mM/day or more, where cysteine is added to the basal medium resulting in the cell culture medium or to the resulting cell culture medium. Preferably, cysteine is added at 0.25 mM/day or more, 0.3 mM/day or more, more preferably 0.4 mM/day or more, more preferably 0.5 mM/day or more. In one embodiment, cysteine is added from 0.225 mM/day to about 0.6 mM/day, from about 0.25 mM/day to about 0.6 mM/day, from about 0.3 mM/day to about 0.6 mM/day, or from about 0.4 mM/day to about 0.6 mM/day.

Without wanting to be bound by theory, cysteine is important in protein synthesis and in the synthesis of glutathione (
Glutathione is used for the production of glutathione (GSH). Glutathione acts as an important intracellular antioxidant that maintains intracellular redox balance by scavenging reactive oxygen species (ROS). ROS are chemically reactive and are by-products of oxygen metabolism. During oxidative stress, ROS levels increase, enhancing RNA and protein damage and promoting apoptosis. Thus, the addition of cysteine can maintain redox balance through the reduction of oxidative stress, which may lead to higher survival rates.

The greater availability of lactate may result in the preferred consumption of lactate as an alternative source of pyruvate instead of glucose. Lactate is highly produced in the early stages of cell culture, and the metabolic shift from lactate production to lactate consumption in cell cultures, especially in high-density or very high-density cell cultures, is approximately at day 3 of culture. From approximately day 5 onwards, lactate may be limiting, especially in high-density or very high-density cell cultures, if not supplemented. It is believed that the consumption of large amounts of lactate results in a lower input to glycolysis and therefore to TCA. This, together with the reduced production of reactive oxygen species, affects the entire metabolism. The combination of lactate and cysteine may be beneficial, since they partly target the same effectors. Cysteine affects the antioxidant pathway of glutathione, together with the reduced levels of reactive oxygen species, and lactate downregulates the metabolism and therefore the production of ROS.

The method according to the invention is a method of culturing cells in vitro, which includes the use of a mammalian cell line, which is used for high expression of a product of interest, such as a heterologous protein or a recombinant virus. Thus, in one embodiment, the mammalian cell is a mammalian cell line, preferably an immortalized cell line. Preferred examples of mammalian cells or mammalian cell lines are CHO cells (e.g. DG44 and K1), NS0 cells, HEK293 cells (e.g. HEK293 cells, HEK293F cells, and HEK293T cells) and BHK21 cells. Preferably, the mammalian cells or mammalian cell lines are adapted to grow in suspension. In a preferred embodiment, the mammalian cells or mammalian cell lines are CHO cells. In a particular embodiment, the mammalian cells are HEK293 cells or CHO cells, or cells derived from HEK293 cells or CHO cells, preferably the mammalian cells are CHO cells or CHO-derived cells.

The term "mammalian cells" as used herein refers to a mammalian cell line suitable for the production of a product of interest, such as a heterologous protein or a recombinant virus, which may also be referred to as a "host cell". Mammalian cells are preferably transformed and/or immortalized cell lines. They are adapted to continuous passage in cell culture, preferably in serum-free cell culture and/or preferably as suspension cultures, and do not include primary untransformed cells or cells that are part of an organ structure. Preferred mammalian cells for the production of heterologous proteins are rodent cells, such as hamster cells, in particular BHK21 cells, BHK TK-cells, CHO cells, CHO-K1 cells, CHO-DXB11 cells (also referred to as CHO-DUKX cells or DuxB11 cells), CHO-S cells and CHO-DG44 cells, or derivatives/progenies of any of such cell lines. Particularly preferred are CHO cells, such as CHO-DG44 cells, CHO-K1 cells, and BHK21 cells, and even more preferred are CHO-DG44 cells and CHO-K1 cells. Most preferred are CHO-DG44 cells. Mammalian cells, particularly glutamine synthase (GS)-deficient derivatives of CHO-DG44 cells and CHO-K1 cells, are also included. These cells are particularly suitable for glutamine synthase-based selection (e.g., methionine sulfoximine (MSX) selection) of clones stably expressing heterologous proteins. In one embodiment of the invention, the mammalian cells are Chinese hamster ovary (CHO) cells, preferably CHO-DG44 cells, CHO-K1 cells, CHO DXB11 cells, CHO-S cells, CHO GS-deficient cells, or derivatives thereof.

Preferred mammalian cells for the production of recombinant viruses are hamster or human cells, in particular BHK21 cells, BHK TK-cells, CHO cells (also called CHO-K1 cells, CHO-DXB11 cells (also called CHO-DUKX cells or DuxB11 cells, CHO-S cells and CHO-DG44 cells) and HEK293 cells, or derivatives/progeny of any of such cell lines. More preferably, the mammalian cells for the production of recombinant viruses are human cells, preferably adapted to serum-free suspension culture, e.g. HEK293 cells and their derivatives (including HEK293F cells and HEK293T cells).

The mammalian cells may further comprise one or more expression cassette(s) encoding a heterologous protein, e.g., a therapeutic protein, preferably a secreted recombinant therapeutic protein. The host cells may also be murine cells, e.g., murine myeloma cells, e.g., NS0 cells and Sp2/0 cells, or any derivative/descendant of such cell lines. Non-limiting examples of mammalian cells that may be used in the context of the present invention are also summarized in Table 1. However, derivatives/descendants of such cells, other mammalian cells, including but not limited to human, mouse, rat, monkey, and rodent cell lines, may also be used in the present invention, particularly for the production of biopharmaceutical proteins.

Mammalian cells are most preferred when established, adapted and cultured completely under serum-free conditions and optionally in media that do not contain any proteins/peptides of animal origin. Commercially available media such as Ham F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Improved Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S (Invitrogen), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any of the media may be supplemented with various compounds as needed, non-limiting examples of which are recombinant hormones and/or other recombinant growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose, or other equivalent energy sources, antibiotics, and trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. For the growth and selection of genetically modified cells expressing a selection gene, an appropriate selection substance is added to the culture medium.

As used herein, the term "heterologous protein" refers to any protein that is not naturally expressed by a mammalian cell and that is introduced into a mammal using recombinant techniques. Preferably, a recombinant nucleic acid is introduced into the mammalian cell, for example by transfection or transduction. The nucleic acid may be stably integrated into the genome or expressed transiently. Preferably, the nucleic acid encoding the heterologous protein is stably integrated into the genome. Preferred mammalian cell lines for expression of heterologous proteins are CHO cells, such as CHO-DG44 cells and CHO-K1 cells.

The heterologous protein can be any therapeutically relevant protein. Examples of therapeutic proteins are, but are not limited to, antibodies, fusion proteins, cytokines, and growth factors. Heterologous proteins produced in mammalian cells according to the methods of the present invention include, but are not limited to, antibodies or fusion proteins, such as Fc-fusion proteins. Other heterologous proteins can be, for example, enzymes, cytokines, lymphokines, adhesion molecules, receptors, and derivatives or fragments thereof, as well as any other polypeptides and scaffolds that can act as agonists or antagonists and/or have therapeutic or diagnostic uses.

A preferred heterologous protein is an antibody or a fragment or derivative thereof, or a fusion protein. Thus, the method according to the invention may advantageously be used for the production of an antibody, preferably a monoclonal antibody. Typically, an antibody is monospecific, but an antibody may also be multispecific. Thus, the method according to the invention may be used for the production of a monospecific antibody, a multispecific antibody, or a fragment thereof, preferably an antibody (monospecific), a bispecific antibody, a trispecific antibody, or a fragment thereof, preferably an antigen-binding fragment thereof. Unless otherwise specified, the term "antibody" refers to a monospecific antibody. Exemplary antibodies within the scope of the present invention include, but are not limited to, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD44v6 antibodies, anti-CD49d antibodies, anti-CD52 antibodies, anti-EGFR1 antibodies (HER1), anti-EGFR2 antibodies (HER2), anti-GD3 antibodies, anti-IGF antibodies, anti-VEGF antibodies, anti-TNFα antibodies, anti-IL2 antibodies, anti-IL-5R antibodies, or anti-IgE antibodies, preferably selected from the group consisting of anti-CD20 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD52 antibodies, anti-HER2/neu antibodies (erbB2), anti-EGFR antibodies, anti-IGF antibodies, anti-VEGF antibodies, anti-TNFα antibodies, anti-IL2 antibodies, and anti-IgE antibodies.

The terms "antibody", "antibodies" or "immunoglobulin(s)" as used herein refer to a protein selected from the globulins that are formed naturally from differentiated B lymphocytes (plasma cells) as a response of the host organism to a foreign substance (= antigen). There are various classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG4 antibody. The terms immunoglobulin and antibody are used synonymously herein. Antibodies include monoclonal, monospecific and multispecific (e.g. bispecific or trispecific) antibodies, single chain antibodies, antigen-binding fragments of antibodies (e.g. Fab fragments or F(ab')2 fragments), disulfide-linked Fvs, etc. The antibody may be of any species, including chimeric and humanized antibodies. A "chimeric" antibody is a molecule in which domains or regions of the antibody are derived from different species. For example, the variable regions of the heavy and light chains may be derived from a rat or mouse antibody, and the constant regions may be derived from a human antibody. In a "humanized" antibody, only minimal sequences are derived from the non-human species. Often, only the CDR amino acid residues of a human antibody are replaced with CDR amino acid residues from a non-human species, such as mouse, rat, rabbit, or llama. Sometimes, some critical framework amino acid residues that affect antigen binding specificity and affinity are also replaced with non-human amino acid residues. Antibodies may be produced through chemical synthesis, via recombinant or transgenic means, via cell (e.g., hybridoma) culture, or by other means.

Typically, antibodies are tetrameric polypeptides composed of two pairs of heterodimers, each formed by a heavy chain and a light chain. Stabilization of both the heterodimer and the tetrameric polypeptide structures occurs via interchain disulfide bridges. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", the terms "domain" and "region" being used synonymously here. Each domain contains about 70-110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain a "variable domain" or "variable region" at their N-terminus, which has less conserved sequences involved in antigen recognition and binding. The variable region of the light chain is also called "VL" and the variable region of the heavy chain is also called "VH".

Antigen-binding fragments include, but are not limited to, "Fab fragments" (Fragment Antigen-binding = Fab), which consist of the variable regions of both chains linked together by adjacent constant regions. They can be formed from conventional antibodies by protease digestion, for example with papain, but Fab fragments can also be produced by genetic engineering as well. Further antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

Using genetic engineering techniques, it is possible to generate shortened antibody fragments consisting only of the variable region of the heavy chain (VH) and the variable region of the light chain (VL). These are called Fv fragments (variable fragment = fragment of the variable part). These Fv fragments are often stabilized, since they lack the covalent bond between the two chains by the cysteines of the constant chains. It is advantageous to link the variable region of the heavy chain and the variable region of the light chain by a short peptide fragment, for example 10 to 30 amino acids, preferably 15 amino acids. In this way, one peptide chain is obtained, consisting of VH and VL, linked by a peptide linker. This type of antibody protein is known as single chain Fv (scFv). Examples of scFv antibody proteins are known to the skilled person. Antibody fragments and antigen-binding fragments therefore further include Fv fragments and especially scFv.

In recent years, various strategies have been developed to prepare scFv as multimeric derivatives. This is aimed in particular at resulting in recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as increased avidity. To achieve multimerization of scFv, scFv have been prepared as fusion proteins with multimerization domains. The multimerization domains can be, for example, the CH3 region of IgG or coiled-coil structures, such as leucine zipper domains. However, there are also strategies that use the interaction between the VH/VL regions of scFv for multimerization (e.g. diabodies, tribodies and pentabodies). By diabody the skilled person means a bivalent homodimeric scFv derivative. By shortening the linker in the scFv molecule to 5-10 amino acids, homodimers are formed, in which interchain VH/VL superposition occurs. Diabodies can be further stabilized by the incorporation of disulfide bridges. Examples of diabody-antibody proteins are known from the prior art.

By minibody, the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as a dimerization region connected to the scFv via a hinge region (for example also derived from IgG1) and a linker region. Examples of minibody antibody proteins are known from the prior art.

By tribody, the skilled artisan means a trivalent homotrimeric scFv derivative, in which the VH-VL are directly fused without a linker sequence, resulting in the formation of a trimer.

The skilled artisan will also be familiar with the so-called mini-antibodies, which have a bivalent, trivalent or tetravalent structure and are derived from scFv. Multimerization is achieved by dimeric, trimeric or tetrameric coiled-coil structures. In a preferred embodiment of the invention, the gene of interest encodes any of such desired polypeptides as mentioned above, preferably a monoclonal antibody, a derivative thereof or a fragment thereof.

Immunoglobulin fragments consisting of the CH2 and CH3 domains of the antibody heavy chain are called "Fc fragments", "Fc regions" or "Fc" due to their crystallization properties (Fc = crystallizable fragment). They can be formed from conventional antibodies by protease digestion, for example with papain or pepsin, but can also be produced by genetic engineering. The N-terminal part of the Fc fragment can vary depending on how many amino acids of the hinge region are still present.

Antibodies that contain an antigen-binding fragment and an Fc region may also be referred to as full-length antibodies. Full-length antibodies may be monospecific and multispecific, e.g., bispecific or trispecific, antibodies.

Preferred therapeutic antibodies according to the invention are multispecific antibodies, in particular bispecific or trispecific antibodies. Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g. malignant B cells) and effector cells (e.g. T cells, NK cells, or macrophages) in one molecule. Exemplary bispecific antibodies are, but are not limited to, diabodies, BiTE (bispecific T cell-inducing antibody) formats, and DART (dual affinity retargeting) formats. The diabody format segregates cognate variable domains of heavy and light chains with two antigen-binding specificities on two separate polypeptide chains, where the two polypeptide chains are non-covalently linked. The DART format is based on the diabody format, but it provides additional stabilization through a C-terminal disulfide bridge. Trispecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. They can be constructed with bispecific antibody technology, which reconstitutes the antigen-recognition domains of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been made that target CD38 on cancer cells and CD3 and CD28 on T cells. It is particularly difficult to make multispecific antibodies with high product quality.

Another preferred therapeutic protein is a fusion protein, such as an Fc-fusion protein. Thus, the present invention may be advantageously used for the production of a fusion protein, such as an Fc-fusion protein. Furthermore, the method for increasing protein production according to the present invention may be advantageously used for the production of a fusion protein, such as an Fc-fusion protein.

The effector portion of the fusion protein may be the complete sequence or any part of the sequence of a native or modified heterologous protein. The immunoglobulin constant domain sequences may be derived from any immunoglobulin subtype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 subtype or class, e.g., IgG, IgA, IgE, IgD, or IgM. Preferentially, they are derived from human immunoglobulins, more preferably from human IgG, even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc, and scFv fragments linked to the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site. Fc fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct containing, for example, another immunoglobulin domain, an enzymatically active protein moiety or an effector domain. Thus, Fc fusion proteins according to the invention also include single chain Fv fragments linked to the CH2 domain of a heavy chain immunoglobulin constant region containing, for example, an N-linked glycosylation site.

In a further aspect, a method for producing a product of interest is provided using the method of the invention, which further comprises isolating and/or purifying the product of interest, and optionally formulating the product of interest into a pharma- ceutically acceptable formulation. In one embodiment, the product of interest is a heterologous protein or a recombinant virus. In particular, a method for producing a heterologous protein is provided using the method of the invention, which further comprises isolating and/or purifying the heterologous protein, and optionally formulating the heterologous protein into a pharma- ceutically acceptable formulation. Alternatively, a method for producing a recombinant virus is provided using the method of the invention, which further comprises isolating and/or purifying the recombinant virus, and optionally formulating the recombinant virus into a pharma- ceutically acceptable formulation, for example, for vaccination or gene therapy.

The heterologous protein may be a therapeutic protein, particularly an antibody, antibody fragment, antibody derivative, or Fc fusion protein, which is preferably recovered/isolated from the culture medium as a secreted polypeptide. It is necessary to purify the therapeutic protein from other recombinant proteins and host cell proteins to obtain a substantially homogeneous preparation of the heterologous protein. As a first step, cells and/or particulate cell debris are removed from the culture medium. The heterologous protein is further purified from contaminating soluble proteins, polypeptides, and nucleic acids, for example by fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, and chromatography on silica gel or cationic resins, such as DEAE. Methods for the purification of heterologous proteins expressed by mammalian cells are known in the art.

Preferably, the heterologous protein is an antibody or an antigen-binding fragment thereof, a multispecific antibody, such as a bispecific or trispecific antibody, or a multispecific antigen-binding fragment thereof, or a fusion protein. The antibody or multispecific antibody (e.g. a bispecific or trispecific antibody) may be an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody, preferably an IgG1 or IgG4 antibody.

In another embodiment, the product of interest is a recombinant virus. The term "recombinant virus" as used herein refers to any virus produced using recombinant technology, particularly suitable for gene therapy or for modifying cells for adoptive cell transfer. Recombinant viruses can also express modified proteins and/or proteins that are heterologous to the virus. Preferred recombinant viruses include, but are not limited to, lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, reovirus, Newcastle disease virus, measles virus, vaccinia virus, influenza virus, and vesicular stomatitis virus (VSV). Preferably, the recombinant virus is an adeno-associated virus or a vesicular stomatitis virus. A preferred mammalian cell for the production of an adeno-associated virus or a vesicular stomatitis virus is a HEK293 cell or a derivative thereof. For production of recombinant virus, mammalian cells can be stably and/or transiently transfected to contain nucleic acid encoding the recombinant virus, or mammalian cells can be transduced to contain nucleic acid encoding the recombinant virus to efficiently produce the virus. For example, VSV can be produced by transduction of mammalian cells such as HEK293 cells or derivatives thereof in serum-free suspension culture. Thus, in one embodiment, the present invention relates to a method for producing a product of interest in a fed-batch process comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a recombinant virus; b) inoculating a basal medium with the mammalian cells to provide a cell culture; c) adding a feed medium to the cell culture comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium, lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the recombinant virus; and e) optionally isolating the product of interest.

Step (a), which is the step of preparing a mammalian cell containing a nucleic acid encoding a recombinant virus, includes a step of transducing or transfecting the cell to introduce the nucleic acid encoding the recombinant virus, where the transfection can be a transient transfection, a stable transfection, or a combination thereof, where the transfection can include the co-transfection of multiple nucleic acid molecules such as plasmids. In the case of a stably transfected cell containing a nucleic acid encoding a recombinant virus, the mammalian cell containing the nucleic acid encoding the recombinant virus (as well as the stably transfected mammalian cell encoding a heterologous protein) is inoculated into a basal medium to obtain a cell culture, and the transient transfection or transduction can be performed after the cell culture is obtained by inoculation of the mammalian cell inoculated into the basal medium, and even after feeding has begun. Mammalian cells may be transiently transfected with one or more nucleic acid molecules encoding a recombinant virus or transduced with a recombinant virus at a desired cell density, and feeding may be initiated on day 0 or later, immediately after the initiation of cell culture, typically 1, 2 or 3 days after the initiation of culture, which may be before or after transfecting or transducing the mammalian cells. In a preferred embodiment, cells are transiently transfected with one or more nucleic acid molecules encoding a recombinant virus or transduced with a recombinant virus at a desired cell density, either at the time of cell inoculation or after a certain period of time, when the desired cell density is reached (which may be after feeding has commenced, e.g. 1-7 days after the initiation of culture, preferably 2-5 days after the initiation of culture, more preferably 3-5 days after the initiation of culture). Preferably, HEK293 cells or derivatives thereof (e.g., HEK293F cells or HEK293T cells) are transduced with a recombinant virus (e.g., VSV) after inoculation and optionally after feeding, resulting in HEK293 cells or derivatives thereof containing a nucleic acid encoding the recombinant virus (e.g., VSV). Methods for producing recombinant viruses, such as VSV, in serum-free suspension cell cultures are known per se in the art, for example from Elahi S.M. et al., (Journal of Biotechnology (2019) 289:114-149.

In certain other embodiments of the methods of the invention, the nucleic acid encodes a heterologous protein and the product titer and/or specific cellular productivity is increased compared to the product titer and/or specific cellular productivity of a heterologous protein produced by the same method, wherein the feed medium is supplemented with 0.19 mM or less cysteine/day in the absence of lactate, preferably wherein the feed medium is supplemented with less than 0.225 mM cysteine/day in the absence of lactate. In one embodiment, the product titer and/or specific cellular productivity is increased by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, or more than 100%. In one embodiment, the heterologous protein is an antibody or an antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein.

In yet another or additional embodiment, the nucleic acid encodes a heterologous protein, and the relative amount of high mannose structures in the population of heterologous proteins is reduced compared to a population of heterologous proteins produced by the same method, wherein the feed medium is supplemented with 0.19 mM/day or less of cysteine in the absence of lactate, and preferably the feed medium is supplemented with less than 0.225 mM/day of cysteine in the absence of lactate. In one embodiment, the relative amount of high mannose structures in the population of heterologous proteins can be reduced by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, or at least 90%. The high mannose structures can be structures with 5 mannose, structures with 6 mannose, structures with 7 mannose, structures with 8 mannose, and/or structures with 9 mannose. Preferably, the reduced relative amount of high mannose structures in the population of heterologous proteins is a reduced relative amount of structures with 5 mannose in the population of heterologous proteins. In a preferred embodiment, the heterologous protein is an antibody. More preferably, the relative abundance (total) of the population of antibodies having a five-mannose structure is less than 20%, preferably less than 10%, more preferably less than 5%. As used herein, the term "population of heterologous proteins" refers to all heterologous proteins in a sample that are encoded by the same nucleic acid. A population of heterologous proteins may be heterogeneous, for example, with respect to glycosylation or post-translational modification or degradation of individual heterologous proteins in the population.

In yet another or additional embodiment, the nucleic acid encodes a heterologous protein, and the relative amount of acidic species (relative to the total) in the population of heterologous proteins is reduced compared to a population of heterologous proteins produced by the same method, where the feed medium is supplemented with the same concentration of cysteine in the absence of lactate. The relative amount of acidic species in the population of heterologous proteins may be reduced by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, or at least 90%. In one embodiment, the heterologous protein is selected from the group consisting of an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. In a preferred embodiment, the heterologous protein is an antibody (monospecific, bispecific, or trispecific) or an antigen-binding fragment thereof.

In another embodiment of the method according to the invention, the nucleic acid encodes a recombinant virus and the virus titer is increased compared to the virus titer produced by the same method, wherein the feed medium is supplemented with 0.19 mM or less cysteine/day in the absence of lactate, preferably the feed medium is supplemented with less than 0.225 mM cysteine/day in the absence of lactate. In one embodiment, the virus titer is increased by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, or more than 100%.

In a particular embodiment of the method according to the invention, the basal medium is a serum-free and chemically defined medium and the feed medium is a serum-free and chemically defined medium. Furthermore, the basal medium and the feed medium may be protein-free.

a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cell into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture, the feed medium comprising one or more feed supplements, the feed medium comprising a lactate/cysteine molar ratio of about 8:1 to about 50:1 (mmol×L ⁻¹ ×day ⁻¹ /mmol×L−1×day ^{−1 )} to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium. Also provided is a method for reducing acidic species in a heterologous protein produced in a fed-batch process, comprising the steps of: (a) adding lactate and cysteine at least at 0.225 mM/day; d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the heterologous protein, wherein the relative amount (relative to the total) of acidic species in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium has been supplemented with the same concentration of cysteine in the absence of lactate. In one embodiment, the heterologous protein is selected from the group consisting of an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. Preferably, the heterologous protein is an antibody (wherein the antibody can be a monospecific or multispecific antibody) or an antigen-binding fragment thereof.

The relative amount of acidic species in the population of heterologous proteins may be reduced by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, or at least 90%. Preferably, less than 50% of the heterologous proteins in the population are acidic species, more preferably less than 40%, less than 30%, less than 20%, and even more preferably less than 10% of the heterologous proteins in the population are acidic species.

As used herein, the term "acidic species" refers to acidic charged variants of heterologous proteins, particularly recombinant monoclonal antibodies produced by post-translational modification. Acidic species are typically recovered using cation or anion exchange chromatography, e.g., (WCX)-10, and may be characterized by liquid chromatography-mass spectrometry. Acidic charged variants include, but are not limited to, methionine oxidation, asparagine deamination, cysteinylation, glycosylation, and reduced disulfide bonds.

a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cell into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture, the feed medium comprising one or more feed supplements, the feed medium comprising a lactate/cysteine molar ratio of about 8:1 to about 50:1 (mmol×L ⁻¹ ×day ⁻¹ /mmol×L−1×day ^{−1 )} to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium. Also provided is a method for reducing high mannose structures in a heterologous protein produced in a fed-batch process comprising the steps of: d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the heterologous protein, wherein the relative amount of high mannose structures in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium is supplemented with 0.19 mM/day or less of cysteine in the absence of lactate, preferably the feed medium is supplemented with less than 0.225 mM/day of cysteine in the absence of lactate. In a preferred embodiment, the high mannose structure is a five-mannose structure. In one embodiment, the heterologous protein is selected from the group consisting of an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. Preferably, the heterologous protein is an antibody (wherein the antibody may be a monospecific or a multispecific antibody) or an antigen-binding fragment thereof. Also provided herein is a heterologous protein produced by the methods according to the invention, in particular by the methods of reducing high mannose structures in a heterologous protein according to the invention and by the methods of reducing acidic species in a heterologous protein according to the invention.

The term "high mannose structure" as used herein refers to high mannose N-linked glycans that contain terminally unsubstituted mannose sugars. These glycans typically contain 5-9 mannose residues attached to a chitobiose (GlcNAc ₂ ) core and thus include Man ₅ GlcNAc ₂ , Man ₆ GlcNAc ₂ , Man ₇ GlcNAc ₂ , Man ₈ GlcNAc ₂ , and Man ₉ GlcNAc ₂ glycans, which are also referred to as five mannose structures (Man-5), six mannose structures (Man-6), seven mannose structures (Man-7), eight mannose structures (Man-8), and nine mannose structures (Man-9), respectively. High mannose structures are associated with shorter half-lives of heterologous proteins, and structures with five mannose residues are considered representative of high mannose structures and are typically determined to approximate high mannose structures. Thus, the high mannose structure is preferably a structure with five mannose residues.

The term "isolating a product of interest" as used herein includes isolating a cell culture medium containing the product of interest from mammalian cells, and/or purifying the product of interest from the cell culture medium after harvesting the cell culture medium containing the product of interest, and/or lysing the cells and purifying the product of interest from the mammalian cell lysate. Similarly, the term "isolating a heterologous protein" or "isolating an antibody" or "isolating a recombinant virus" as used herein includes isolating a cell culture medium containing a heterologous protein and/or antibody or recombinant virus from mammalian cells, and/or purifying the heterologous protein and/or antibody or recombinant virus from the cell culture medium after harvesting the cell culture medium containing the heterologous protein and/or antibody or recombinant virus, and/or lysing the cells and purifying the heterologous protein and/or antibody or recombinant virus from the mammalian cell lysate. Methods for purifying heterologous proteins, including antibodies or recombinant viruses, are known in the art.

a) providing a mammalian cell comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cell into a basal medium to provide a cell culture; c) adding a feed medium to the cell culture, the feed medium comprising one or more feed supplements, the feed medium comprising a lactate/cysteine molar ratio of about 8:1 to about 50:1 (mmol×L ⁻¹ ×day ⁻¹ /mmol×L−1×day ^{−1 )} to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium. Also provided is a method of preventing the negative effect of cysteine on product quality characteristics when producing heterologous proteins in a fed-batch process, comprising the steps of: (a) adding lactate and cysteine at least at 0.225 mM/day; d) culturing mammalian cells in cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the cell culture medium containing the heterologous protein from the mammalian cells, wherein the negative effect on product quality characteristics of the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium is supplemented with the same concentration of cysteine in the absence of lactate. In one embodiment, the heterologous protein is selected from the group consisting of an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. Preferably, the heterologous protein is an antibody (including a monospecific antibody, a bispecific antibody, or a trispecific antibody) or a fragment thereof. Negative effects on product quality characteristics can be, but are not limited to, an abundance or increased amount of high mannose structures (preferably structures with a high mannose of 5), an abundance or increased amount of low molecular weight species, and/or an abundance or increased amount of acidic species.

Process optimization is especially relevant for heavily seeded bioprocesses. Higher seeding densities minimize the non-productive exponential growth phase, resulting in a relatively short and highly productive process. CHO cells are most commonly used for the production of biopharmaceuticals of heterologous proteins, such as antibodies or fusion proteins, in fed-batch processes. Such processes consist of an initial non-productive or less productive growth phase, where cells accumulate in the bioreactor, followed by a stationary phase, where most of the product is produced. The length of the growth phase directly impacts the duration and volumetric productivity of the process. With the use of a perfusion system in the N-1 seed train, this growth phase is shifted to the N-1 bioreactor. The perfusion mode allows for continuous removal of waste metabolites and addition of nutrients by continuous medium exchange to reach high cell densities of up to 100 x ¹⁰⁶ cells per mL with acceptable viability. Through the application of perfusion process in N-1 stage, high seeding density can be achieved in the subsequent N stage (i.e. fed-batch process), resulting in immediate high productivity. Ultra-high seeding density (uHSD) fed-batch processes can produce the same amount of titer with comparable product quality in a shorter period of time, which improves manufacturing capacity. Furthermore, they can produce higher final product titer in the same length of time than smaller seeding processes. Thus, high seeding density cultures are promising for improving overall productivity, but are also particularly demanding and delicate in terms of medium optimization. To improve the ultra-high seeding density process, the inventors have discovered that cysteine and/or lactate have beneficial effects on the performance of the culture. Surprisingly, this was also found to improve cell cultures using normal seeding density.

According to the present invention, cysteine is added at 0.225 mM/day or more. In particular, in high density seeding processes or ultra-high density seeding processes, it may be beneficial to add cysteine at 0.3 mM/day or more, 0.4 mM/day or more, or 0.5 mM/day or more. In one embodiment, cysteine is added at about 0.3 mM/day to about 0.6 mM/day, or about 0.4 mM/day to about 0.6 mM/day. The increase or decrease according to the present invention can be determined in comparison to a method or culture in which the feed medium is added with 0.19 mM/day or less of cysteine in the absence of lactate, preferably in which the feed medium is added with less than 0.225 mM/day of cysteine in the absence of lactate. In a high density seeding process or an ultra-high density seeding process, when cysteine is added at 0.3 mM/day or more, in one embodiment, the increase or decrease is determined compared to a method or culture in which the feed medium is added with 0.28 mM/day or less of cysteine in the absence of lactate.

However, uHSD processes are often characterized by an early loss of viability. The addition of lactate and cysteine according to the methods of the present invention overcomes these problems. Thus, in one embodiment, the fed-batch process is a uHSD fed-batch process.

Use of lactate and cysteine in a feed medium to improve cell culture performance In one aspect, the present invention relates to the use of lactate in a feed medium to reduce acidic species in a heterologous protein produced in a fed-batch process, wherein the feed medium comprises cysteine. Preferably, the feed medium provides 0.225 mM/day or more of cysteine. In one embodiment of the use of the present invention, the group consists of an antibody or an antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. Preferably, the heterologous protein is an antibody (wherein the antibody can be monospecific and multispecific).

The present invention also relates to the use of lactate in a feed medium to reduce high mannose structures, such as pentamannose structures, in a heterologous protein produced in a fed-batch process, wherein the feed medium comprises cysteine. Preferably, the feed medium provides 0.225 mM cysteine/day or more.

The present invention also relates to the use of cysteine in a feed medium to prevent a negative effect of cysteine on a heterologous protein product quality characteristic, preferably wherein the negative effect on the product quality characteristic is increased high mannose structures (e.g. structures with five mannose), increased low molecular weight species, and/or increased acidic species. In one embodiment, the feed medium provides cysteine at 0.225 mM/day or more, e.g. 0.25 mM/day or more, 0.3 mM/day or more, or 0.4 mM/day or more.

The present invention also relates to the use of lactate and cysteine in a feed medium to increase the titer of a heterologous protein and/or the specific productivity of cells in a fed-batch process. It also provides the use of lactate and cysteine in a feed medium to increase recombinant virus production in a fed-batch process.

In one embodiment of the use of the present invention, the heterologous protein is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. Preferably, the heterologous protein is an antibody (wherein the antibody can be monospecific and multispecific). The fed-batch process according to the use of the present invention comprises culturing a mammalian cell, where the mammalian cell can be any mammalian cell as described herein, preferably the mammalian cell is a HEK293 cell or a CHO cell, or a cell derived from a HEK293 cell or a CHO cell, preferably the mammalian cell is a CHO cell or a CHO-derived cell.

The use of lactate or lactate and cysteine is according to the method of the present invention. Thus, lactate and cysteine are to be added at a molar ratio of lactate/cysteine of about 8:1 to 50:1, about 10:1 to 50:1, preferably about 10:1 to about 30:1, more preferably about 15:1 to about 30:1, even more preferably about 15:1 to about 30:1. In one embodiment, lactate is added at 3 mmol/L/day or more, 3.8 mmol/L/day or more, 5 mmol/L/day or more, preferably 7 mmol/L/day or more of lactate, 7.8 mmol/L/day or more, 10 mmol/L/day or more, or even 15 mmol/L/day or more. Lactate in the cell culture medium can be maintained at 0.5 g/L or more, 1 g/L or more, preferably 2 g/L or more, preferably 2-4 g/L, more preferably about 2-3 g/L. More specifically, the concentration of lactate in the cell culture medium is maintained at about 1 g/L to about 4.5 g/L (about 10-50 mM), about 2 g/L to about 4 g/L (about 20-45 mM), preferably about 2 g/L to about 3 g/L (about 20-35 mM). Lactate (molecular weight = 89.07 g/mol) may be provided as its salt and/or hydrate and/or lactic acid, preferably as sodium lactate (molecular weight = 112.06 g/mol), where 1 g/L of lactate is equivalent to about 1.25 g/L of sodium lactate. The lactate salt and/or hydrate or lactic acid is provided in equimolar concentrations to the lactate concentrations provided herein. In a preferred embodiment, the basal medium does not contain lactate added as a medium component. However, lactate may be produced in the basal medium as a metabolite during the culture.

Cysteine may be provided as cysteine or its salts and/or hydrates, cystine or its salts, or a dipeptide or tripeptide containing cysteine. The salts and/or hydrates of cysteine, or cystine or its salts, or a dipeptide or tripeptide containing cysteine are provided in equimolar concentrations to the concentrations of cysteine provided herein. According to the present invention, cysteine is added at 0.225 mM/day or more. Cysteine is added to the basal medium that gives rise to the cell culture medium or to the resulting cell culture medium. Preferably, cysteine is added at 0.25 mM/day or more, 0.3 mM/day or more, more preferably 0.4 mM/day or more, more preferably 0.5 mM/day or more. In one embodiment, cysteine is added from about 0.225 mM/day to about 0.6 mM/day, from about 0.25 mM/day to about 0.6 mM/day, from about 0.3 mM/day to about 0.6 mM/day, or from about 0.4 mM/day to about 0.6 mM/day.

In a particular embodiment of the use according to the invention, the heterologous protein product titer and/or the specific cell productivity is increased compared to the heterologous protein product titer and/or the specific cell productivity produced by the same method, wherein the feed medium is supplemented with 0.19 mM cysteine/day or less in the absence of lactate, preferably wherein the feed medium is supplemented with less than 0.225 mM cysteine/day in the absence of lactate. In one embodiment, the product titer and/or the specific cell productivity is increased by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, or more than 100%.

In another embodiment, the relative amount of high mannose structures in the heterologous protein population may be reduced by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, or at least 90%. Reduced means compared to a heterologous protein population produced by the same method, where the feed medium is supplemented with 0.19 mM/day or less of cysteine in the absence of lactate, and preferably where the feed medium is supplemented with less than 0.225 mM/day of cysteine in the absence of lactate. The high mannose structures may be structures with 5 mannose, structures with 6 mannose, structures with 7 mannose, structures with 8 mannose, and/or structures with 9 mannose. Preferably, the reduced relative amount of high mannose structures in the heterologous protein population is a reduced relative amount of structures with 5 mannose in the heterologous protein population. In a preferred embodiment, the heterologous protein is an antibody, and the relative amount (total) of the population of antibodies having a structure with five mannose residues is less than 20%, preferably less than 10%, more preferably less than 5%, or even less than 2%.

In yet another or additional embodiment, the heterologous protein may be selected from the group consisting of an antibody or antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein, and the relative amount of acidic species (relative to the total) in the population of heterologous proteins is reduced compared to a population of antibodies produced by the same method, where the feed medium is supplemented with the same concentration of cysteine in the absence of lactate. The relative amount of acidic species in the population of antibodies may be reduced by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, or at least 90%. Preferably, the heterologous protein is an antibody (wherein the antibody may be monospecific or multispecific).

In another embodiment of the use according to the invention, a recombinant virus is produced and the virus titer is increased compared to the virus titer produced by the same method, wherein the feed medium is supplemented with 0.19 mM or less cysteine/day in the absence of lactate, preferably wherein the feed medium is supplemented with less than 0.225 mM cysteine/day in the absence of lactate. In one embodiment, the virus titer is increased by at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, or more than 100%.

The present invention may also be used in high seeding density and ultra-high seeding density (uHSD) fed-batch processes as described herein.

Feed medium for improved cell culture performance In yet another aspect, the present invention relates to a feed medium for mammalian cell fed-batch culture comprising lactate and cysteine in a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1, wherein cysteine is added at 0.225 mM/day or more. Preferably, the feed medium comprises one or more feed supplements for separate addition. In particular, cysteine may be added in a two-feed strategy, for example adding a feed medium containing cysteine and a separately added feed supplement containing cysteine. Lactate is preferably added with the feed medium, but may also be added separately in a feed supplement. In a particular embodiment, the feed medium or feed supplement is chemically defined (optionally containing a recombinant protein such as insulin or insulin-like growth factor (IGF)). Furthermore, the feed medium or feed supplement according to the invention does not contain cells (i.e. is not a cell culture medium containing cells), is not in contact with cells in a culture medium (pre-conditioned medium obtained from a cell culture medium) and/or does not contain metabolic waste products derived from cells. The feed medium according to the invention can be used in the methods and uses described herein, and therefore the embodiments set out and described for the methods apply to the feed medium as well.

The feed medium may also be provided in a kit. Thus, the present invention also relates to a kit comprising (a) a concentrated feed medium for a mammalian cell fed-batch culture, comprising lactate and optionally cysteine, and (b) an aqueous supplementary substance isolated from the concentrated feed medium comprising cysteine, wherein the feed medium and supplementary substance provide a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1 and 0.225 mM/day or more of cysteine, at a daily addition of less than 5%, preferably less than 4%, more preferably less than 3.5% of the starting cell culture volume. The feed medium provided by the kit according to the present invention may be used in the methods and uses described herein, and therefore the embodiments set out and described for the methods apply to the kit as well.

In particular, the feed medium and kits are particularly useful for fed-batch processes involving culturing mammalian cells, where the mammalian cells can be any mammalian cell as described herein, preferably the mammalian cells are HEK293 cells or CHO cells, or cells derived from HEK293 cells or CHO cells, preferably the mammalian cells are CHO cells or CHO-derived cells.

The feed medium is adapted to provide lactate and cysteine according to the method of the present invention. Thus, the feed medium or the kit for providing the feed medium is adapted to provide lactate and cysteine in a molar ratio of lactate/cysteine of about 8:1 to 50:1, about 10:1 to 50:1, preferably about 10:1 to about 30:1, more preferably about 15:1 to about 30:1, even more preferably about 15:1 to about 25:1. In one embodiment, lactate is provided at 3 mmol/L/day or more, 3.8 mmol/L/day or more, 5 mmol/L/day or more, preferably 7 mmol/L/day or more, 7.8 mmol/L/day or more, 10 mmol/L/day or more, or even 15 mmol/L/day or more. The lactate salt (molecular weight = 89.07 g/mol) may be provided as its salts, esters, and/or hydrates, and/or as lactic acid, preferably as a salt, e.g., sodium lactate (molecular weight = 112.06 g/mol), where 1 g/L of lactate salt is equivalent to about 1.25 g/L of sodium lactate. The lactate salts and/or hydrates or lactic acid are provided in equimolar concentrations to the lactate salt concentrations provided herein.

Cysteine may be provided in the feed medium or kit according to the invention as cysteine or its salts and/or hydrates, cystine or its salts, or a dipeptide or tripeptide containing cysteine. The salts and/or hydrates of cysteine, or cystine or its salts, or a dipeptide or tripeptide containing cysteine are provided in equimolar concentrations to the concentrations of cysteine provided herein. The feed medium or kit is adapted to provide cysteine to the basal medium or cell culture medium at 0.225 mM/day or more. Preferably, cysteine is added at 0.25 mM/day or more, 0.3 mM/day or more, more preferably 0.4 mM/day or more, more preferably 0.5 mM/day or more. In one embodiment, cysteine is added from about 0.225 mM/day to about 0.6 mM/day, from about 0.25 mM/day to about 0.6 mM/day, from about 0.3 mM/day to about 0.6 mM/day, or from about 0.4 mM/day to about 0.6 mM/day.

The feed media and kits described herein may also be used in high seeding density and ultra-high seeding density (uHSD) fed-batch processes as described herein.

In view of the above, it will be appreciated that the present invention also encompasses the following:

Item 1 provides a method for producing a product of interest in a fed-batch process, comprising: a) providing mammalian cells comprising a nucleic acid encoding the product of interest; b) inoculating a basal medium with the mammalian cells to provide a cell culture; c) adding a feed medium to the cell culture, comprising adding one or more feed supplements, wherein the feed medium is a basal medium resulting in a cell culture medium, or a resulting cell culture medium, to which lactate and cysteine are added in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest; and e) optionally isolating the product of interest.

Item 2 provides a method according to item 1, in which the feed medium is added once a day, preferably continuously.

Item 3 provides a method according to item 1 or 2, wherein the lactate/cysteine molar ratio is about 10:1 to 50:1, preferably about 10:1 to about 30:1.

Item 4 provides the method according to any one of items 1 to 3, wherein lactate is added at 3 mmol/L/day or more, 5 mmol/L/day or more, 7 mmol/L/day or more, or 10 mmol/L/day or more.

Item 5 provides a method according to item 4, wherein lactate in the cell culture medium is maintained at 0.5 g/L or more, 1 g/L, 2 g/L or more, preferably 2-4 g/L.

Item 6 provides a method according to any one of items 1 to 5, wherein cysteine (a) is provided as cysteine or a salt and/or hydrate thereof, cystine or a salt thereof, or a dipeptide or tripeptide containing cysteine, and/or (b) cysteine is added at 0.25 mM/day or more, 0.3 mM/day or more, or 0.4 mM/day or more.

Item 7 provides the method of any one of items 1 to 6, wherein the product of interest is a heterologous protein or a recombinant virus.

Item 8 provides the method of any one of items 1 to 7, wherein the nucleic acid encodes a heterologous protein, and the product titer and/or specific productivity of the cells is increased compared to the product titer and/or specific productivity of the cells of the heterologous protein produced by the same method, and wherein the feed medium is supplemented with 0.19 mM/day or less of cysteine in the absence of lactate.

Item 9 provides the method of any one of items 1 to 8, wherein the nucleic acid encodes a heterologous protein, and the relative amount of high mannose structures in the population of heterologous proteins is reduced compared to a population of heterologous proteins produced by the same method, wherein the feed medium is supplemented with 0.19 mM or less cysteine per day in the absence of lactate, and preferably wherein the high mannose structures are structures having 5 mannoses.

Item 10 provides the method of any one of items 1 to 9, wherein the nucleic acid encodes a heterologous protein, and the relative amount (relative to the total) of acidic species in the population of heterologous proteins is reduced compared to a population of heterologous proteins produced by the same method, wherein the feed medium is supplemented with the same concentration of cysteine in the absence of lactate.

Item 11 provides the method according to any one of items 1 to 10, wherein the basal medium and the feed medium are serum-free and have a defined chemical composition.

Item 12 provides the method of any one of items 1 to 11, wherein the heterologous protein is an antibody or an antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein.

Item 13 provides the method of item 12, wherein the antibody, bispecific antibody, or trispecific antibody is an IgG1 antibody, an IgG2a antibody, an IgG2b antibody, an IgG3 antibody, or an IgG4 antibody.

Item 14 provides a method for culturing mammalian cells in a fed-batch process, comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a product of interest; b) inoculating a basal medium with the mammalian cells to provide a cell culture; c) adding a feed medium to the cell culture, comprising adding one or more feed supplements, wherein the feed medium is added to the basal medium resulting in the cell culture medium, or to the resulting cell culture medium, with lactate and cysteine in a molar ratio of about 8:1 to about 50:1 lactate/cysteine (mmol×L ⁻¹ ×day ⁻¹ /mmol×L ⁻¹ ×day ⁻¹ ), wherein cysteine is added at 0.225 mM/day or greater; and d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest.

Item 15 provides a method for producing a heterologous protein comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cells into a basal medium to provide a cell culture; and c) adding a feed medium to the cell culture, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium or to the resulting cell culture medium, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium comprising adding ^one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium comprising ^{adding one} or more feed ^supplements to the resulting ... a) adding lactate and cysteine, wherein cysteine is added at 0.225 mM/day or more; d) culturing mammalian cells in a cell culture medium under conditions that permit expression of the heterologous protein; and e) optionally isolating the heterologous protein; wherein the relative amount of acidic species in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium has been supplemented with the same concentration of cysteine in the absence of lactate.

Item 16 provides a method for producing a heterologous protein comprising the steps of: a) providing mammalian cells comprising a nucleic acid encoding a heterologous protein; b) inoculating the mammalian cells into a basal medium to provide a cell culture; and c) adding a feed medium to the cell culture, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium or to the resulting cell culture medium, the feed medium comprising adding one or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium ^comprising adding one ^or more feed supplements to the basal medium resulting in the cell culture medium, the feed medium ^comprising adding one or more feed supplements to the resulting ^... a) adding lactate and cysteine at or above 0.225 mM/day; d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the heterologous protein, wherein the relative amount of high mannose structures in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium is added with 0.19 mM/day or less of cysteine in the absence of lactate, and preferably wherein the high mannose structures are structures with 5 mannose units.

Item 17 provides a method for producing a heterologous protein comprising the steps of: (a) providing mammalian cells comprising a nucleic acid encoding a heterologous protein; (b) inoculating the mammalian cells into a basal medium to provide a cell culture; and (c) adding one or more feed supplements to the cell culture, the feed medium comprising a lactate/cysteine molar ratio of about 8:1 to about 50:1 (mmol×L−1×day−1/mmol×L ^{− 1} ×day ⁻¹ ) to the basal medium resulting in the cell culture medium or to the ^resulting cell culture medium. (d) culturing mammalian cells in a cell culture medium under conditions that permit expression of the heterologous protein; and (e) optionally isolating the heterologous protein from the mammalian cells, wherein the negative effect of cysteine on product quality characteristics in the population of heterologous protein is reduced compared to a population of heterologous protein produced by the same method, wherein the feed medium has been supplemented with the same concentration of cysteine in the absence of lactate.

Item 18 provides the method of any one of items 1 to 17, wherein the mammalian cell is a HEK293 cell or a CHO cell, or a cell derived from a HEK293 cell or a CHO cell, preferably the mammalian cell is a CHO cell or a CHO-derived cell.

Item 19 provides a heterologous protein produced by the method of any one of items 15 to 17.

Item 20 provides the use of lactate in a feed medium to reduce acidic species in a heterologous protein produced in a fed-batch process, wherein the feed medium is supplemented with cysteine at 0.225 mM/day or more.

Item 21 provides the use of lactate in a feed medium to reduce high mannose structures in a heterologous protein produced in a fed-batch process, wherein the feed medium contains cysteine at 0.225 mM/day or more, and preferably wherein the high mannose structures are structures having 5 mannoses.

Item 22 provides the use of lactate in a feed medium to prevent the negative effects of cysteine on product quality characteristics of a heterologous protein produced in a fed-batch process, preferably wherein the negative effects on product quality characteristics are increased high mannose structures, decreased low molecular weight species, and/or increased acidic species.

Item 23 provides the use of lactate and cysteine in a feed medium to increase the titer of a heterologous protein and/or the specific productivity of cells in a fed-batch process.

Item 24 provides the use of any one of items 20 to 23, wherein the fed-batch process comprises culturing mammalian cells, wherein the mammalian cells are HEK293 cells or CHO cells, or cells derived from HEK293 cells or CHO cells, preferably the mammalian cells are CHO cells or CHO-derived cells.

Item 25 provides a feed medium for mammalian cell fed-batch culture comprising lactate and cysteine in a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1.

Item 26 provides the feed medium of item 25, wherein the feed medium includes one or more feed supplements for separate addition.

Item 27 provides a kit including (a) a concentrated feed medium for mammalian cell fed-batch culture containing lactate and optionally cysteine, and (b) an aqueous supplement isolated from the concentrated feed medium containing cysteine, wherein the feed medium and supplement provide a lactate/cysteine molar ratio (mM/mM) of about 8:1 to about 50:1 and 0.225 mM/day or more of cysteine, with daily addition of less than 5%, preferably less than 3.5%, of the cell culture starting volume.

EXAMPLES Example 1: Ultra-High Seeding Density (uHSD) Process A Chinese Hamster Ovary (CHO) cell line (cell line A; CHO-K1 GS) producing a monoclonal IgG1 antibody (mAb) was cultivated in a 3 L glass bioreactor system in fed-batch mode. The seed train culture was processed in shake flasks up to the N-1 stage, which was processed in a 2 L disposable bioreactor system in perfusion mode. The cell seeding density was set at 10 x 106 cells/ml in a proprietary basal medium containing 0.4 g/L cysteine HCl _H2O (2.3 mM cysteine; molecular weight of cysteine HCl _H2O = 173.63 g/mol), ^0.028 g/L L-cystine 2HCl (0.36 mM cysteine; molecular weight of cystine HCl = 157.62 mM) and no lactate in a starting volume of 2.2 L. Fed-batch cultivation was carried out for 13-14 days. Feed medium (1.21 g/L cysteine HCl H ₂ O; 0.0069 M cysteine, no lactose) was added continuously from day 0 to the end of the process (days 0-9; 45 ml/L/day; days 9-14; 25 ml/L/day), glucose was added to the process on demand to maintain above 3 g/L to 5 g/L. Sodium lactate and additional cysteine HCl H ₂ O were added as bolus additions as shown in Table 2. Lactate was added to the bioprocesses uHSD LAC and uHSD LAC/CYS by bolus additions from day 3 to day 13 when the lactate concentration dropped below 2 g/L with a target concentration of 3 g/L (stock solution 238.5 g/L; 2.68 mol/L). Addition of a bolus of cysteine to the processes uHSD LAC/CYS and uHSD/CYS was performed from day 1 to day 5 in a volume of 7 ml (stock solution cysteine HCl H ₂ O: 30 g/L (20.69 g/L cysteine; 0.17 mol/L). Culture samples were taken every 24 h and cell counts and cell viability determinations were performed using a Cedex HiRes analyzer (Roche, Germany). Glucose and lactate were determined using a Konelab Prime60i (Thermo Scientific, USA). Antibody concentrations were determined using a Protein A HPLC method (Thermo Scientific, USA).

The effect of lactose, cysteine, or lactose and cysteine on viable cell density (VCD), viability, product concentration, and lactate concentration is shown in Figure 1A-D.

As can be seen from Figures 1A and B, feeding with lactate and cysteine (uHSD LAC/CYS) and feeding with cysteine only (uHSD CYS) improved viability and viable cell density starting from about day 6 compared to cells fed without additional lactate and cysteine feed (uHSD) and cells fed with lactate only (uHSD LAC). Notably, towards the end of the culture, the viability of cells fed with lactate and cysteine (uHSD LAC/CYS) appears to be even higher than cells fed with cysteine only (uHSD CYS).

Regarding production, lactate feeding (uHSD LAC) had a positive effect on IgG titers during the culture, but this did not seem to be sustainable until the end of the culture. IgG titers in cell cultures receiving an additional cysteine feed (uHSD CYS) were comparable to control cell cultures (uHSD). Surprisingly, IgG titers in cell cultures containing lactate and cysteine (uHSD LAC/CYS cells) were strongly increased compared to bolus feeds containing lactate or cysteine only. This was mainly due to the increased specific productivity (pg/cell/day) from day 10 onwards (data not shown).

As shown in Figure 1D, lactate in the cultures was depleted between approximately days 5 and 10 and was persistently greater than 2 g/L in uHSD LAC/CYS cultures. The decrease in lactate in uHSD CYS cultures on days 6 and 9 is likely due to the high cell concentration and high specific uptake rate of lactate.

At high overall density feeding, the uHSD process with bolus addition of sodium lactate and cysteine resulted in improved product titer and cell viability profiles.

Example 2: Experimental design optimization study in a routine fed-batch process To further analyze the effect of cysteine and/or lactate on the cell culture medium in more detail, we performed a routine fed-batch process using a routine seeding density with two different cell lines under controlled conditions using a bioreactor.

Two CHO-K1 GS cell lines (cell line A and B) producing IgG1 monoclonal antibody (mAb) were cultivated in an Amber 250 bioreactor system, respectively. The experiments were part of a Design of Experiments (DoE) study. The seed train cultures were processed in shake flasks and the seeding cell density was set at ^0.7x106 cells/ml. The fed-batch cultivation was carried out for 14 days. In contrast to the uHSD process, continuous application of lactate and cystine was applied in these experiments to reduce the high concentration of the reactive compound cysteine in the bioreactor and lactate was included in the routinely applied feed.

Feed medium (1.1 g/L cysteine HCl H ₂ O; 0.0063 M cysteine) was added continuously from day 2 at 30 ml/L/day (of culture starting volume) until the end of the process, and glucose was added to the process on demand. Culture samples were taken every 24 hours and cell counts and cell viability determinations were performed using a Cedex HiRes analyzer (Roche, Germany). Glucose and lactate were determined using a Konelab Prime60i (Thermo Scientific, USA). Antibody concentrations were determined using a Protein A HPLC method (Thermo Scientific, USA). Charge variants were analyzed using a PrpPacWCX-10 analytical column connected to an HPLC system with a UV detector. Size variants were analyzed using a BEH200 size exclusion chromatography column connected to an HPLC system with a UV detector. N-glycan determination was performed using the LabChip GXII and HT Glycan Reagent Kit.

A Design of Experiments (DoE) study was conducted to identify the interactions of feeding various concentrations of cysteine and lactate. A DoE study is a data collection and analysis tool that allows for the variation of multiple input factors and determines their combined and independent effects on various output parameters. Thus, this type of study can identify the interactions of multiple factors in a process by simultaneously varying the levels of multiple inputs in the process.

The experimental design test was based on an I-optimal design, which included a feeding range of 0 to 1.67 ml/L/day (i.e., 0, 0.84, and 1.67 ml/L/day, which correspond to 0.12 and 0.24 mM/day) with the factor cystine (as a second feed with 17.2 g/L cystine (equivalent to approximately 143 mM cysteine)) and sodium lactate (included in the routine feed at 30 ml/L/day) in a stock solution of 0 to 30 g/L (i.e., 0, 15, or 30 g/L, which correspond to 0, 0.133, and 0.267 M, added once daily at 0, 4, and 8 mM/day). Cysteine was added with the feed medium (6.3 mM at 30 ml/L/day; equivalent to a daily addition of 0.19 mM/day), so the total daily addition of cysteine in samples designated 0, 0.84, and 1.64 is 0.19, 0.31, and 0.43 mM/day, respectively.

The effect of cysteine and/or lactate feeding on viable cell density, viability, and product titer, and lactate concentration in a routine process for cell line A is shown in Figures 2A-D, and for cell line B in Figures 2E-H. For both cell lines, beneficial effects on titer (Figures 2C and G) and cell viability (Figures B and F) were observed with lactate and cystine feeds.

The positive effect of feeding a combination of lactate and cystine on harvest viability and product titer at harvest is presented in the contour plots of the experimental design (cell line A: Figures 3 and 4; cell line B: Figures 6 and 7). Higher product titers were achieved using cell line B, and the product titers in Figures 4 and 7 are presented as normalized [%] to the highest titer in the experiment, i.e., cell line B. The use of various concentrations for cell line B showed that the highest product titers could be obtained with high amounts of lactate and high amounts of cysteine. Similar results were shown for cell line A, again demonstrating that high amounts of lactate and high amounts of cysteine increase harvest viability and product titer.

Cysteine, as a known antioxidant, is also known to increase the acidic charge variants of antibodies. Therefore, the effect of lactate and cysteine feeding combination on product quality attributes such as acidic charge variants (acidic peak group (APG)), low molecular weight species (LMW), and high mannose species was determined. Surprisingly, a positive effect of lactate feeding on APG, LMW and high mannose structures was demonstrated, which can be seen from Figures 5 and 8 (APG), 9 (high mannose) and 10 (LMW).

Figures 5 and 8 show the APG for cell lines A and B as a function of lactate and cystine feeding, respectively. As can be seen from Figures 5 and 8, the increase in APG due to cysteine feeding can be strongly reduced through additional lactate feeding. Furthermore, as can be seen from Figures 9A and B, the pentamannose structure (Man5) of the antibody can be reduced with increasing lactate concentrations for two different IgG1 antibodies produced by cell lines A and B. Finally, Figure 10 shows the LMW normalized to the highest value of the experimental design (obtained with cell line B) for cell lines A and B as a function of cysteine (A and C) and lactate (B and D) concentrations. As can be seen from Figure 10, the LMW of the produced antibodies was reduced with increasing lactate or cystine feeding, resulting in a synergistic effect in which the LMW was reduced with both high lactate and high cysteine concentrations.

Example 3: Reproducibility of results with additional cell lines Four additional CHO cell lines producing monoclonal antibodies (mAbs), including CHO-DG44 GS cells and CHO-K1 GS cells, were cultured in the Amber 15 bioreactor system. Cell lines C and F each produce an IgG4 monoclonal antibody with a different specificity, and cell lines D and E each produce an IgG1 monoclonal antibody with a different specificity. The seed train cultures were processed in shake flasks and the seeding cell density was set at ^0.7x106 cells/ml. The fed-batch cultures were run for 14 days. Feed medium was added continuously from day 2 until the end of the process, and glucose was added to the process on demand as described in Example 2. Furthermore, the process was carried out with variable feeding using a feed medium containing cysteine (1.1 g/L cysteine HCL _H2O ; 0.0063M) and lactate (267 mM) or a feed medium containing cysteine but no lactate (added as a routine continuous feed with a culture starting volume of 30 ml/L/day, feed 1) and/or a second cysteine feed (feed 2) as shown in Table 3. Lactate was obtained as sodium lactate or as lactic acid, which was titrated with NaOH to sodium lactate before being added to the feed medium. Culture samples were taken every 24 hours and cell counts and cell viability determinations were performed using a Cedex HiRes analyzer (Roche, Germany). Glucose, lactate, and antibody concentrations were determined using a Konelab Prime60i (Thermo Fisher Scientific, USA) or a Biosen S-line (EKF Diagnostics GmbH, UK).

Experiments with four additional CHO cell lines confirmed the positive effect of the lactate and cystine feeding combination on process performance. The highest cell viability and product titers were obtained with the lactate and cystine feeding combination in all four cell lines (see Figures 11-14).

Example 4: Experimental design optimization of the uHSD process Two CHO-K1 GS cell lines A and B producing IgG1 monoclonal antibody (mAb) were cultivated in an Amber 250 bioreactor system. The seed train culture was processed in shake flasks up to the N-1 stage, which was processed in a 2 L disposable bioreactor system in perfusion mode. The fed-batch cultivation was carried out for 14 days. Feed medium (1.1 g/L cysteine HCl H ₂ O (0.0063 M cysteine) and 0, 15 or 30 g/L sodium lactate (0, 0.133 or 0.267 M lactate) were added continuously from day 0 to the end of the process. The seeding cell density (SCD) was set at 5-10×10 ⁶ cells/ml. Glucose was added to the process on demand. Culture samples were taken every 24 h and cell counts and cell viability determinations were performed using a Cedex HiRes analyzer (Roche, Germany). Glucose and lactate were determined using a Konelab Prime60i (Thermo Scientific, USA). Antibody concentrations were determined using a Protein A HPLC method (Thermo Scientific, USA).

The design of experiments (DoE) study was based on an I-optimal design, including factors cystine (as a second feed with 5.98 g/L cystine (equivalent to approximately 49.83 mM)) in the feeding range of 0-4.8 ml/L/day (i.e., 0, 2.4, and 4.8 ml/L/day, corresponding to 0, 0.12, and 0.24 mM/day), and sodium lactate (included in the routine feed at 45 ml/L/day from days 0 to 9 and at 25 ml/L/day from days 9 to 14 of the culture starting volume) of 0-30 g/L (i.e., 0, 15, or 30 g/L; corresponding to 0, 0.133, and 0.267 M, respectively, with 5.98 and 11.96 mM/day at 45 ml/L/day, and 3.32 and 6.64 mM/day at 25 ml/L/day, added once daily). Cysteine was also added with the feed medium (at 6.3 mM at 45 ml/L/day; equivalent to a daily addition of 0.28 mM/day, and at 25 ml/L/day; equivalent to a daily addition of 0.16 mM/day), so the total daily additions of cysteine in samples added with 0, 2.4, and 4.8 ml/L/day in the second feed are 0.28, 0.4, and 0.52 mM/day at 45 ml/L/day once a day, and 0.16, 0.28, and 0.4 mM/day at 25 ml/L/day once a day.

The positive effect of feeding a combination of lactate and cystine on product titer is presented in the contour plots of the experimental design in Figure 15 for cell line A and Figure 16 for cell line B at harvest day 14. Higher product titers were achieved using cell line B, and the product titers in Figures 15 and 16 are presented as the highest titer in the experiment, normalized [%] to cell line B. The highest product titers were obtained with high lactate feeding and high cysteine feeding for both cell lines tested.

The positive correlation between harvest viability and lactate feeding is further presented in Figures 17 and 18. The post-fit R2 and Q2 predictive performance are presented for each model. The effect on product quality attributes, such as acidic charge variants and high mannose, at harvest is presented in Figures 19 to 22. It is shown that for both cell lines, the increase in acidic charge variants (APGs) due to high cysteine feeding was strongly reduced by additional lactate feeding.

Claims (18)

a) providing a mammalian cell containing a nucleic acid encoding a product of interest;
b) inoculating mammalian cells into a basal medium to provide a cell culture;
c) adding a feed medium to the cell culture comprising lactate and cysteine or adding one or more feed supplements comprising lactate and/or cysteine together or separately with a feed medium comprising lactate and/or cysteine or both to the cell culture, whereby lactate and cysteine are added to the cell culture medium in a molar ratio of lactate/cysteine (mM/day/mM/day) of about 8:1 to about 50:1, and cysteine is added at 0.225 mM/day or greater;
d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the product of interest; and e) optionally isolating the product of interest. The method of claim 1, wherein the lactate/cysteine molar ratio is about 10:1 to 50:1. The method of claim 2, wherein the lactate/cysteine molar ratio is from about 10:1 to about 30:1. a) in the step of adding a feed medium to the cell culture, lactate is added at 3 mM/day or more, 5 mM/day or more, 7 mM/day or more, or 10 mM/day or more;
b) lactate in the cell culture medium is maintained at 0.5 g/L or more, 1 g/L or more, or 2 g/L or more;
4. The method according to claim 1, wherein c) in the step of adding the feed medium to the cell culture, cysteine is provided as cysteine or a salt and/or hydrate thereof, cystine or a salt thereof, or a di- or tripeptide containing cysteine; and/or d) in the step of adding the feed medium to the cell culture, cysteine is added at 0.25 mM/day or more, 0.3 mM/day or more, or 0.4 mM/day or more. 5. The method of claim 4, wherein lactate in the cell culture medium is maintained at 2-4 g/L. The method of any one of claims 1 to 5, wherein the product of interest is a heterologous protein or a recombinant virus. the nucleic acid encodes a heterologous protein, and (a) the product titer and/or specific cellular productivity is increased compared to the product titer and/or specific cellular productivity of the heterologous protein produced by the same method, differing only in that the cell culture medium is supplemented with 0.19 mM/day or less of cysteine and no lactate in the step of adding a feed medium to the cell culture;
(b) the relative amount (%) of heterologous proteins having high mannose structures in the produced population of heterologous proteins is reduced compared to the relative amount (%) of heterologous proteins having high mannose structures in the population of heterologous proteins produced by the same method, the only difference being that 0.19 mM/day or less of cysteine and no lactate is added to the cell culture medium in the step of adding a feed medium to the cell culture medium; and/or (c) the relative amount (%) of acidic species in the produced population of heterologous proteins is reduced compared to the relative amount (%) of acidic species in the population of heterologous proteins produced by the same method, the only difference being that no lactate is added to the cell culture medium in the step of adding a feed medium to the cell culture medium.
The method according to any one of claims 1 to 6. The method of claim 7, wherein the high mannose structure is a structure having five mannose units. The method according to any one of claims 1 to 8, wherein the basal medium and the feed medium are serum-free and have a defined chemical composition. The method according to any one of claims 1 to 9, wherein the heterologous protein is an antibody or an antigen-binding fragment thereof, a bispecific antibody, a trispecific antibody, or a fusion protein. a) providing a mammalian cell containing a nucleic acid encoding a product of interest;
b) inoculating mammalian cells into a basal medium to provide a cell culture;
c) adding a feed medium to the cell culture comprising lactate and cysteine, or adding one or more feed supplements comprising lactate and/or cysteine together or separately with a feed medium containing lactate and/or cysteine or not containing both, to the cell culture, whereby lactate and cysteine are added to the cell culture medium in a molar ratio of lactate/cysteine (mM/day/mM/day) of about 8:1 to about 50:1, and cysteine is added at 0.225 mM/day or greater; and d) culturing the mammalian cells in the cell culture medium under conditions that allow expression of the product of interest. The method of claim 11, wherein the product of interest is a heterologous protein or a recombinant virus. a) providing a mammalian cell containing a nucleic acid encoding a heterologous protein;
b) inoculating a basal medium with mammalian cells to provide a cell culture;
c) adding a feed medium to the cell culture comprising lactate and cysteine or adding one or more feed supplements comprising lactate and/or cysteine together or separately with a feed medium comprising lactate and/or cysteine or both to the cell culture, whereby lactate and cysteine are added to the cell culture medium in a molar ratio of lactate/cysteine (mM/day/mM/day) of about 8:1 to about 50:1, and cysteine is added at 0.225 mM/day or greater;
d) culturing mammalian cells in a cell culture medium under conditions that allow expression of the heterologous protein; and e) optionally isolating the heterologous protein, wherein the relative amount (%) of acidic species in the population of heterologous protein produced in a fed-batch process is reduced compared to the relative amount (%) of acidic species in the population of heterologous protein produced by the same process, differing only in that no lactate is added to the cell culture medium in the step of adding a feed medium to the cell culture. a) providing a mammalian cell containing a nucleic acid encoding a heterologous protein;
b) inoculating mammalian cells into a basal medium to provide a cell culture;
c) adding a feed medium to the cell culture comprising lactate and cysteine or adding one or more feed supplements comprising lactate and/or cysteine together or separately with a feed medium comprising lactate and/or cysteine or both to the cell culture, whereby lactate and cysteine are added to the cell culture medium in a molar ratio of lactate/cysteine (mM/day/mM/day) of about 8:1 to about 50:1, and cysteine is added at 0.225 mM/day or greater;
A method for reducing high mannose structures in a heterologous protein produced in a fed-batch process, comprising the steps of: d) culturing mammalian cells in a cell culture medium under conditions allowing expression of the heterologous protein; and e) optionally isolating the heterologous protein, wherein the relative amount (%) of heterologous protein having high mannose structures in the population of produced heterologous proteins is reduced compared to the relative amount (%) of heterologous protein having high mannose structures in the population of heterologous proteins produced by the same method, the only difference being that in the step of adding a feed medium to the cell culture medium, 0.19 mM/day or less of cysteine and no lactate are added to the cell culture medium. The method of claim 14, wherein the high mannose structure is a structure having five mannose units. a) providing a mammalian cell containing a nucleic acid encoding a heterologous protein;
b) inoculating mammalian cells into a basal medium to provide a cell culture;
c) adding a feed medium to the cell culture comprising lactate and cysteine or adding one or more feed supplements comprising lactate and/or cysteine together or separately with a feed medium comprising lactate and/or cysteine or both to the cell culture, whereby lactate/cysteine is added to the cell culture medium at a molar ratio of lactate/cysteine (mM/day/mM/day) of about 8:1 to about 50:1, and cysteine is added at 0.225 mM/day or greater;
d) culturing mammalian cells in a cell culture medium under conditions allowing expression of the heterologous protein; and e) optionally isolating the heterologous protein from the mammalian cells, wherein the negative effect of cysteine on product quality characteristics in a population of heterologous proteins is reduced compared to a population of heterologous proteins produced by the same process differing only in that no lactate is added to the cell culture medium in the step of adding a feed medium to the cell culture, and the negative effect on product quality characteristics is increased high mannose structures, increased low molecular weight species, and/or increased acidic species. The method of any one of claims 1 to 16, wherein the fed-batch process includes a step of culturing mammalian cells, the mammalian cells being HEK293 cells or CHO cells, or cells derived from HEK293 cells or CHO cells. 18. The method of claim 17, wherein the mammalian cell is a CHO cell or a CHO-derived cell .

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